Type VI collagen mutations in Bethlem myopathy, an autosomal dominant myopathy with contractures.

Among the diverse family of collagens, the widely expressed microfibrillar type VI collagen is believed to play a role in bridging cells with the extracellular matrix. Several observations imply substrate properties for cell attachment as well as association with major collagen fibers. Previously, we have established genetic linkage between the genes encoding the three constituent alpha-chains of type VI collagen and Bethlem myopathy. A distinctive feature of this autosomal dominant disorder consists of contractures of multiple joints in addition to generalized muscular weakness and wasting. Nine kindreds show genetic linkage to the COL6A1-COL6A2 cluster on chromosome 21q22.3 (refs 3,4; manuscript submitted) whereas one family shows linkage to markers on chromosome 2q37 close to COL6A3 (ref. 5). Sequence analysis in four families reveals a mutation in COL6A1 in one and a COL6A2 mutation in two other kindreds. Both mutations disrupt the Gly-X-Y motif of the triple helical domain by substitution of Gly for either Val or Ser. Analogous to the putative perturbation of the anchoring function of the dystrophin-associated complex in congenital muscular dystrophy with mutations in the alpha 2-subunit of laminin, our observations suggest a similar mechanism in Bethlem myopathy.

Genetic localization of Bethlem myopathy.

Bethlem myopathy is a rare autosomal dominant myopathy characterized by slowly progressive limb-girdle muscular atrophy and weakness, and contractures of multiple joints. To identify the genetic localization we used highly polymorphic microsatellite markers in a genome-wide search in six Dutch families. After excluding genetic linkage with 52 markers distributed evenly over the autosomes, significant linkage was present with the 21q22.3 locus PFKL (two-point lod score of Zmax = 6.86 at theta = 0.03). There was no indication of genetic heterogeneity. The pattern of recombinations observed with adjacent markers indicated a localization distal to PFKL. Recombination of a marker within the collagen 6a1 gene (COL6A1) excluded this apparent candidate gene in one of the Bethlem myopathy families. The disease gene is most likely located in the region between COL6A1 and the telomere of chromosome 21q.

Differential diagnosis in spinal and bulbar muscular atrophy clinical and molecular aspects.

Kennedy disease is caused by an enlarged trinucleotide repeat sequence within the androgen receptor gene. We report here seven male patients with a benign motor neuron syndrome highly analogous to Kennedy disease but with a normal trinucleotide repeat.

Identification of a two base pair deletion in five unrelated families with adrenoleukodystrophy: a possible hot spot for mutations.

The gene for X-linked adrenoleukodystrophy (ALD) was recently identified. Intragenic deletions of several kilobases were found in about 7% of patients. Point mutations, expected to be very heterogeneous, were identified so far in only two patients. We report the identification of a two base pair deletion at position 1801-1802 of the ALD cDNA, located within the fifth exon of the ALD gene, which precedes the two consensus motives for ATP-binding. This microdeletion was found in five out of 40 unrelated ALD kindreds, indicating that this position is a hot spot for mutations. The mutation was observed both in patients with childhood cerebral ALD (CCALD) and in patients with adrenomyeloneuropathy (AMN).

Deletion of the serine 34 codon from the major peripheral myelin protein P0 gene in Charcot-Marie-Tooth disease type 1B.

Charcot-Marie-Tooth disease type 1B (CMT1B) is genetically linked to chromosome 1q21-23. The major peripheral myelin protein gene, P0, has been cloned and localized to the same chromosomal region. P0 is a 28 kDa glycoprotein involved in the compaction of the multilamellar myelin sheet and accounts for more than half of the peripheral myelin protein content. We checked whether P0 is altered in CMT1B, and show here that a 3 basepair deletion in exon 2 of the P0 gene is present in all affected individuals of a CMT1B family. The mutation results in the deletion of serine 34 in the extracellular domain of P0, suggesting that alterations of P0 cause CMT1B.

Differentiation and proliferation of respiration-deficient human myoblasts.

Replication and transcription of mitochondrial DNA were impaired in dividing human myoblasts exposed to ethidium bromide. MtDNA content decreased linearly per cell division and mitochondrial transcript levels declined rapidly, resulting in respiration-deficiency of the myoblasts. Despite the absence of functional mitochondria the cells remained able to proliferate when grown under specific culture conditions. However, the formation of myotubes was severely impaired in respiration-deficient myoblasts. We conclude that differentiation of myoblasts into myotubes is more dependent on mitochondrial function than proliferation of myoblasts.

Identical point mutations of PMP-22 in Trembler-J mouse and Charcot-Marie-Tooth disease type 1A.

We have investigated the peripheral myelin protein gene, PMP-22, in a family with Charcot-Marie-Tooth disease type 1A (CMT1A). The DNA duplication commonly found in CMT1A was absent in this family, but strong linkage existed between the disease and the CMT1A marker VAW409R3 on chromosome 17p11.2. We found a point mutation in PMP-22 which was completely linked with the disease. The mutation, a proline for leucine substitution in the first putative transmembrane domain, is identical to that recently found in the Trembler-J mouse. The presence of this PMP-22 defect in this CMT1A family and the location of PMP-22 within the DNA duplication associated with CMT1A suggest that both structural alteration and overexpression of PMP-22 may lead to the disease.

Metallothionein in Menkes' disease: induction in cultured muscle cells.

Menkes' disease is an inherited disturbance of copper metabolism. Addition of copper to the medium of cultured fibroblasts and lymphoblasts from patients with Menkes' disease results in an increased induction of metallothionein. We investigated the metallothionein induction in response to copper and zinc in muscle cells (myoblasts and myotubes). Metallothionein synthesis was analyzed by gel electrophoresis of labeled proteins and metallothionein synthesis in muscle cells was compared with the synthesis in fibroblasts. The induction by copper was higher both in muscle cells and in fibroblasts from the Menkes' patient compared to the control cells. Hybrid myotubes obtained by fusion of control myoblasts and Menkes' myoblasts render a system in which complementation can be studied. Metallothionein synthesis in hybrid myotubes occurred at a level intermediate between the synthesis in Menkes' and control myotubes. The abnormal accumulation of copper-induced metallothionein was only partially corrected by fusion with normal cells. Metallothionein induction by zinc was similar in Menkes' and control fibroblasts. Combination of copper and zinc yielded no differences in additional metallothionein synthesis for Menkes' cells and control fibroblasts. Therefore, metallothionein induction in Menkes' disease can primarily be accounted for by copper rather than by zinc.

Distribution and characterization of a Sandhoff disease-associated 50-kb deletion in the gene encoding the human beta-hexosaminidase beta-chain.

A 50-kb deletion was demonstrated in the gene encoding for the beta-subunit of human hexosaminidase (HEXB), using field inversion gel electrophoresis (FIGE) of SfiI-digested chromosomal DNA from patients with Sandhoff disease. We investigated 14 patients from different parts of Europe and found no deletion in 5 patients, 2 patients homozygous for the deletion, and 7 patients with the deletion in one allele. The distribution of the 50-kb deletion was approximately in agreement with the Hardy-Weinberg equilibrium. The deletion was characterized using chromosomal DNA from one of the two homozygous patients. Restriction fragments were hybridized with a 1.6-kb (almost complete) and a 0.4-kb (5') HEXB cDNA clone. It appeared that the deletion started in intron 5, extending in the 5' direction and causing the loss of exon 1-5 and the promoter area of the HEXB gene.

Demonstration of a Sandhoff disease-associated autosomal 50-kb deletion by field inversion gel electrophoresis.

Field inversion gel electrophoresis (FIGE) of SfiI-digested chromosomal DNA was used to demonstrate a 50-kb deletion in one allele of the gene encoding the beta subunit of human hexosaminidase (HEXB at 5q13) of two apparently unrelated patients with Sandhoff disease. In conventional electrophoretic restriction analysis, this deletion was masked by hybridization of bands from the other allele.

Prospect for enzyme therapy in glycogenosis II variants: a study on cultured muscle cells.

Impairment of skeletal muscle function is the common feature of distinct clinical forms of glycogenosis type II. In the present study, muscle cultures from different patients were used to investigate the cause of clinical heterogeneity and the feasibility of enzyme replacement therapy. The activity of acid alpha-glucosidase appears to be the primary factor in determining the extent of lysosomal glycogen storage in muscle, and thereby the clinical severity of the disease. Neutral alpha-glucosidases do not seem influential. Correction of the enzymatic defect was achieved in skeletal muscle cultures from patients by administration of a "high-uptake" form of acid alpha-glucosidase, purified from human urine. The enzyme reaches the lysosomes, including the glycogen storage vacuoles, and the lysosomal glycogen content is reduced to control level. In normal muscle cells 20% of the total cellular glycogen pool is segregated in lysosomal compartments. This percentage is higher than in fibroblasts, which may partly explain why muscles are more prone to store glycogen. The relevance of this study for enzyme therapy is discussed.

Collagen synthesis in cultured myoblasts and myotubes from patients with Duchenne muscular dystrophy.

Collagen synthesis was studied in cultured myoblasts and myotubes from 4 patients with Duchenne muscular dystrophy (DMD) and 4 control persons. Incorporation of [3H]proline into collagen of DMD cells and control cells was not significantly different. The same types of collagen, i.e., type I and type III were synthesized by myogenic cells from DMD patients and controls. In contrast to others, we could not obtain evidence for an increase in the degree of prolyl hydroxylation in collagen of the DMD muscle cells.

Uptake and stability of human and bovine acid alpha-glucosidase in cultured fibroblasts and skeletal muscle cells from glycogenosis type II patients.

Acid alpha-glucosidase (EC 3.2.1.20) was purified from human placenta and bovine testis by affinity chromatography using concanavalin A (conA) and Sephadex G 200. When added to the culture medium of human fibroblasts, the enzyme purified from bovine testis is taken up with a 200-fold higher efficiency than the enzyme from human placenta. Uptake of acid alpha-glucosidase from bovine testis is mediated by the mannose-6-phosphate receptor, whereas only a minor fraction of placental enzyme appears to be equipped with the mannose-6-phosphate recognition marker. Once internalized, both human and bovine acid alpha-glucosidase demonstrate a half-life of about 10 days in fibroblasts from control individuals and patients with different clinical forms of glycogenosis type II (Pompe's disease, acid alpha-glucosidase deficiency). Evidence is presented that the mannose-6-phosphate receptor is also present on the plasma membrane of the clonal myogenic skeletal muscle cell lines G8-1 and L6J1 (respectively from mouse and rat origin) and on cultured human skeletal muscle cells derived from a muscle biopsy. Addition of bovine testis acid alpha-glucosidase to skeletal muscle cell cultures from an adult patient with glycogenosis type II leads to complete correction of the enzyme deficiency.