Functional studies on in situ-like mitochondria isolated in the presence of polyvinyl pyrrolidone.

Mitochondria isolated and maintained in sucrose mannitol medium show a large intermembrane space and a condensed matrix unlike the appearance of in situ mitochondria. Mitochondria resembling in situ organelles are obtained when the isolation medium is supplemented with certain macromolecules such as polyvinyl pyrrolidone. We found that the in situ appearance was acquired also by the conventionally isolated mitochondria when they were exposed to 2% polyvinyl pyrrolidone supplemented medium. Paradoxically, however, these in situ looking mitochondria proved functionally inferior in that their brief incubation without substrates led to a marked loss of their ability to respire with subsequently added substrates such as pyruvate, acylcarnitines or glutamate. The oxidation of succinate was, however, not so affected. This phenomenon was shared by heart and skeletal muscle mitochondria of different animal species but not by rat liver mitochondria. The inhibition of respiration could not be related to the failure to oxidize NADH, to the tieing up of mitochondrial free CoASH, or to the increased matrix space of mitochondria that was observed in the presence of polyvinyl pyrrolidone. The polyvinyl pyrrolidone-exposed mitochondria regained their respiratory ability on being freed from polyvinyl pyrrolidone. The same phenomenon was seen also when the medium contained 2% albumin or 20% Ficoll.

Identical dysferlin mutation in limb-girdle muscular dystrophy type 2B and distal myopathy.

Limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM) are autosomal recessive disorders caused by mutations in the dysferlin gene on chromosome 2p13. The authors studied a large Russian family with both LGMD2B and MM. All affected individuals, as well as one preclinical boy with dystrophic changes on muscle biopsy, were found to be homozygous for a novel dysferlin mutation, TG573/574AT (Val67Asp). This finding supports the view that additional factors (e.g., modifier genes) contribute to the phenotypic expression of causative mutations in dysferlinopathies.

Dysferlin expression after normal myoblast transplantation in SCID and in SJL mice.

Limb girdle muscular dystrophy type 2B form and Miyoshi myopathy are both caused by mutations in the recently cloned gene dysferlin. In the present study, we have investigated whether cell transplantation could permit dysferlin expression in vivo. Two transplantation models were used: SCID mice transplanted with normal human myoblasts, and SJL mice, the mouse model for limb girdle muscular dystrophy type 2B and Miyoshi myopathy, transplanted with allogeneic primary mouse muscle cell cultures expressing the beta-galactosidase gene under control of a muscle promoter of Troponin I. FK506 immunosuppression was used in the non-compatible allogeneic model. One month after transplantation, human and mouse dysferlin proteins were detected in all transplanted SCID and SJL muscles, respectively. Co-localization of dysferlin and human dystrophin or beta-galactosidase-positive fibers was observed following the transplantation of myoblasts. Dysferlin proteins were monitored by immunocytochemistry and Western blot. The number of dysferlin-positive fibers was 40-50% and 20-30% in SCID and SJL muscle sections, respectively. Detection of dysferlin in both SCID mice and dysferlin-deficient SJL mouse shows that myoblast transplantation permits the expression of the donor dysferlin protein.

Secondary reduction in calpain 3 expression in patients with limb girdle muscular dystrophy type 2B and Miyoshi myopathy (primary dysferlinopathies).

Dysferlin is the protein product of the gene (DYSF) that is defective in patients with limb girdle muscular dystrophy type 2B and Miyoshi myopathy. Calpain 3 is the muscle-specific member of the calcium activated neutral protease family and primary mutations in the CAPN3 gene cause limb girdle muscular dystrophy type 2A. The functions of both proteins remain speculative. Here we report a secondary reduction in calpain 3 expression in eight out of 16 patients with a primary dysferlinopathy and clinical features characteristic of limb girdle muscular dystrophy type 2B or Miyoshi myopathy. Previously CAPN3 analysis had been undertaken in three of these patients and two showed seemingly innocuous missense mutations, changing calpain 3 amino acids to those present in the sequences of calpains 1 and 2. These results suggest that there may be an association between dysferlin and calpain 3, and further analysis of both genes may elucidate a novel functional interaction. In addition, an association was found between prominent expression of smaller forms of the 80 kDa fragment of laminin alpha 2 chain (merosin) and dysferlin-deficiency.

The 56 kDa androgen-binding protein in human genital skin fibroblasts: its relation to the human androgen receptor.

We have recently described in genital skin fibroblasts (GSF) a relatively abundant 56 kDa protein with androgen-binding activity. This protein is missing in GSF of most patients with complete androgen insensitivity syndrome (CAI). The protein has many characteristics compatible with the androgen receptor; it has in fact been tentatively considered as a precursor or degradation form of the prototypic (approximately 100 kDa) human androgen receptor. We have prepared an antiserum to this protein, which allowed us to detect it as a direct product by in vitro translation of mRNA from GSF. It is thus very unlikely to be a degradation product of a larger precursor. Furthermore, covalent photolytic labeling of this protein with the androgen analogue [3H]mibolerone revealed a much lower affinity for this protein than is known for the androgen receptor. Finally, the GSF of two exceptional patients with complete androgen insensitivity syndrome due to negligible androgen receptor-binding activity express this protein normally, as determined on two-dimensional gels by Western blot analysis with the antiserum and by photolytic covalent labeling with androgen analogues. These data indicate that the protein is not a precursor or a degradation product of the receptor; nor is it androgen-induced. They are more compatible with the idea that the protein is another member of the steroid/thyroid/retinoic acid receptor supergene family, perhaps as an unorthodox product of the human androgen receptor gene.

The 56/58 kDa androgen-binding protein in male genital skin fibroblasts with a deleted androgen receptor gene.

Human genital skin fibroblasts (GSF) make a relatively abundant 56/58 kDa protein that binds androgens. The protein shares many properties with the approximately 100 kDa androgen receptor that is encoded by a locus in the q12 region of the X chromosome. It does not appear to be androgen-induced, yet is absent in GSF of most patients with complete androgen insensitivity (CAI). A precursor-product relation with the androgen receptor (AR) protein has been largely excluded; that it may be an unorthodox product of the AR gene has not. The 56/58 kDa protein is made by the GSF of a mentally retarded subject who has CAI because of a complete deletion of the coding portion of the AR gene. Hence, the strong constitutional and statistical correlations that have been demonstrated between the two proteins cannot arise because they share the same gene. The subject's genomic DNA hybridizes normally with 11 single-copy probes from Xq11-Xq13. Therefore, we cannot attribute her mental retardation to a contiguous gene syndrome.

Three years' experience with neonatal screening for Duchenne/Becker muscular dystrophy: gene analysis, gene expression, and phenotype prediction.

Neonatal screening for Duchenne/Becker Muscular dystrophy (DMD/BMD) was begun as a pilot program on January 1, 1986. The aim of this program was to reduce the incidence of this X-linked recessive degenerative neuromuscular disease. The neonatal detection of a boy with DMD allows early identification of carriers and genetic counselling. This may avert the birth of other affected males born prior to clinical diagnosis of DMD in the propositus at about age 5 years. Between January 1, 1986, and December 31, 1988, we identified and characterized a cohort of 8 asymptomatic infant boys with grossly elevated levels of creatine kinase, an active primary dystrophic process of muscle and complete dystrophin deficiency. Five of 8 males have detectable DNA alterations involving the DMD/BMD locus. Based on current hypotheses, characterization of dystrophin expression of this cohort allows us to predict a DMD phenotype in all 8 boys. To date, no additional males with DMD have been born in these families. Prospective follow-up will allow us to test the validity of dystrophin testing in predicting the clinical course and impact of this program on reproductive decision making in these families.

Amber mutation creates a diagnostic MaeI site in the androgen receptor gene of a family with complete androgen insensitivity.

We have discovered in the X-linked androgen receptor gene a single nucleotide substitution that is the putative cause of complete androgen insensitivity (resistance) in a family with affected individuals in 2 generations. Earlier studies on the family indicated co-segregation of mutant phenotype and the RFLPs at the loci DXS1 and DXYS1. The mutation is an adenine-to-thymine transversion in exon 8 that changes the sense of codon 882 from lysine to an amber (UAG) translation termination signal. The substitution creates a recognition sequence for the restriction endonuclease MaeI: this permits ready recognition of hemizygotes and heterozygotes after amplification of genomic exon 8 by the polymerase chain reaction. The mutation predicts the synthesis of a truncated receptor that lacks 36 amino acids at the carboxy terminus of its 252-amino acid androgen-binding domain. The cultured genital skin fibroblasts of the one affected patient examined have normal levels of androgen receptor mRNA, but negligible androgen-receptor binding activity. These results accord with a variety of data from spontaneous and artificial mutations indicating that all portions of the steroid binding domain contribute to normal steroid binding by a steroid receptor.

Limb girdle muscular dystrophy in Manitoba Hutterites does not map to any of the known LGMD loci.

Limb girdle muscular dystrophy (LGMD) is a heterogeneous group of disorders affecting primarily the shoulder and pelvic girdles. Autosomal dominant and recessive forms have been identified; 8 have been mapped and 1 more has been postulated on the basis of exclusion of linkage. An autosomal recessive muscular dystrophy was first described in 1976 in the Hutterite Brethren, a North American genetic and religious isolate [Shokeir and Kobrinsky, 1976; Clin Genet 9:197-202]. In this report, we discuss the results of linkage analysis in 4 related Manitoba Hutterite sibships with 21 patients affected with a mild autosomal recessive form of LGMD. Because of the difficulties in assigning a phenotype in some asymptomatic individuals, stringent criteria for the affected phenotype were employed. As a result, 7 asymptomatic relatives with only mildly elevated CK levels were assigned an unknown phenotype to prevent their possible misclassification. Two-point linkage analysis of the disease locus against markers linked to 7 of the known LGMD loci and 3 other candidate genes yielded lod scores of < or = -2 at theta = 0.01 in all cases and in most cases at theta = 0.05. This suggests that there is at least 1 additional locus for LGMD.

Partial androgen insensitivity--are all tissues equal?

Nonaromatizable androgens, administered in high doses to an adult patient with partial androgen insensitivity, failed to result in a change in phallic size despite a clear decline in SHBG and gonadotropin levels. These findings raise the question of differential tissue sensitivity to androgens. Because ancillary laboratory testing does not predict reliably the genital response, a therapeutic trial should be advocated in such cases.

Analysis of the CAG repeat region of the androgen receptor gene in a kindred with X-linked spinal and bulbar muscular atrophy.

Herein we describe a family with X-linked spinal and bulbar muscular atrophy (SBMA or Kennedy's disease), an adult onset neuromuscular disease characterized by slow progression, predominant proximal and bulbar muscle weakness. One frequent association is the appearance of gynecomastia. This disorder was previously shown to be linked to the locus DXYS1 on the proximal long arm of the X chromosome. Recently, a report implicated a mutation at the N-terminus of the androgen receptor gene involving amplification of CAG repeats as the cause of X-linked SBMA. We studied this region of the androgen receptor in a kindred clinically suspected but not confirmed of having X-linked SBMA by the polymerase chain reaction (PCR) followed by Southern analysis and DNA sequencing. The mutated allele was found to have an increased number of 51 CAG repeats confirming the clinical diagnosis of SBMA. Normal individuals revealed 23 repeat numbers within the normal range, while another unrelated X-linked SBMA patient had an enlarged CAG repeat region. The carrier or disease status could be established or confirmed in 12 individuals of this family on the basis of detecting normal and disease alleles reflected by the number of CAG repeats.

Proline incorporation by cultured skin fibroblasts from patients with duchenne muscular dystrophy.

The incorporation of proline by cultured skin fibroblasts derived from normal individuals and patients with Duchenne muscular dystrophy (DMD) was analyzed by SDS-gel electrophoresis combined with a double-labeling procedure [Pediatr. Res., 12: 887 (1978)]. DMD fibroblasts showed increased proline incorporation into protein of approximately 130 000 daltons which could be degraded partially by collagenase. This difference was observed only at 7 days after seeding, and may be due to slight differences in growth rate as comparison of cells harvested at 2 and 7 days indicated that increased proline incorporation into high molecular weight protein was associated with day 7 cells, whether normal or DMD. During 14 days in culture normal and DMD strains did not differ in protein content, doubling time or incorporation of [3H]leucine and [14C]proline into cellular protein, although the ratio of [14C]proline to [3H]leucine incorporated was greater for DMD fibroblasts at 7 days. Incorporation of [14C]proline and [3H]leucine into extracellular proteins was greater in DMD fibroblast cultures. These subtle differences support the hypothesis that the DMD gene is expressed in fibroblasts.

Single-blind study of dystrophin staining in carriers of Duchenne muscular dystrophy.

A single-blind study of dystrophin staining in skeletal muscle was performed in 13 biopsies from carriers of Duchenne Muscular Dystrophy (DMD) and controls. The results indicate that immunohistochemical analysis of dystrophin staining is a valuable diagnostic test for DMD carriers when DNA for testing is unavailable from critical family members or is uninformative, when creatine kinase (CK) values are conflicting or when CK values must be used in isolation.

Mitochondrial calcium content and oxidative phosphorylation in heart and skeletal muscle of dystrophic mice.

Mitochondrial calcium overloading was investigated in the genetically dystrophic mouse (strains 129/ReJ dy/dy) as a possible contributing factor to the development of muscle fiber necrosis. Mitochondrial calcium concentrations were significantly elevated in both skeletal muscle and heart organelles. Because mitochondria were isolated in the presence of ruthenium red this finding was not the result of an artefact of isolation. State 3 respiration rates and concomitantly the respiratory control ratios were slightly decreased in skeletal muscle, but not in heart mitochondria. This abnormality could result from calcium overloading in a small fraction of the mitochondria. Fractionation of skeletal muscle mitochondria on sucrose gradients gave two distinct populations of dystrophic organelles, one with high calcium, whereas normal skeletal muscle mitochondria and heart organelles showed only one broad band on the gradient. The results support the idea that both skeletal muscle and heart are affected in dystrophic mice, strain 129/ReJ dy/dy and also that in the dystrophic mouse the process of cell necrosis is associated with cellular calcium overloading.