Left-right axis malformations associated with mutations in ACVR2B, the gene for human activin receptor type IIB.

Targeted disruption of the mouse activin receptor type IIB gene (Acvr2b) results in abnormal left-right (LR) axis development among Acvr2b-/- homozygotes [Oh and Li, 1997: Genes Dev 11:1812-1826]. The resulting malformations include atrial and ventricular septal defects, right-sided morphology of the left atrium and left lung, and spleen hypoplasia. Based on these results, we hypothesized that mutations in the type IIB activin receptor gene are associated with some cases of LR axis malformations in humans. We report here characterization of the ACVR2B genomic structure, analysis of ACVR2B splice variants, and screening for ACVR2B mutations among 112 sporadic and 14 familial cases of LR axis malformations. Two missense substitutions have been identified, one of which appears in two unrelated individuals. Neither of these nucleotide changes has been found in 200 control chromosomes. We conclude that ACVR2B mutations are present only rarely among human LR axis malformation cases.

X-linked situs abnormalities result from mutations in ZIC3.

Vertebrates position unpaired organs of the chest and abdomen asymmetrically along the left-right (LR) body axis. Each structure comes to lie non-randomly with respect to the midline in an overall position designated situs solitus, exemplified in humans by placement of the heart, stomach and spleen consistently to the left. Aberrant LR axis development can lead to randomization of individual organ position (situs ambiguus) or to mirror-image reversal of all lateralized structures (situs inversus). Previously we mapped a locus for situs abnormalities in humans, HTX1, to Xq26.2 by linkage analysis in a single family (LR1) and by detection of a deletion in an unrelated situs ambiguus male (Family LR2; refs 2,3). From this chromosomal region we have positionally cloned ZIC3, a gene encoding a putative zinc-finger transcription factor. One frameshift, two missense and two nonsense mutations have been identified in familial and sporadic situs ambiguus. The frameshift allele is also associated with situs inversus among some heterozygous females, suggesting that ZIC3 functions in the earliest stages of LR-axis formation. ZIC3, which has not been previously implicated in vertebrate LR-axis development, is the first gene unequivocally associated with human situs abnormalities.

A submicroscopic deletion in Xq26 associated with familial situs ambiguus.

Abnormal left-right-axis formation results in heterotaxy, a multiple-malformation syndrome often characterized by severe heart defects, splenic abnormalities, and gastrointestinal malrotation. Previously we had studied a large family in which a gene for heterotaxy, HTX1, was mapped to a 19-cM region in Xq24-q27.1. Further analysis of this family has revealed two recombinations that place HTX1 between DXS300 and DXS1062, an interval spanning approximately 1.3 Mb in Xq26.2. In order to provide independent confirmation of HTX1 localization, a PCR-based search for submicroscopic deletions in this region was performed in unrelated males with sporadic or familial heterotaxy. A cluster of sequence-tagged sites failed to amplify in an individual who also had a deceased, affected brother. FISH identified the mother as a carrier of the deletion, which arose as a new mutation from the maternal grandfather. The deletion interval spans 600-1,100 kb and lies wholly within the 1.3-Mb region identified by recombination. Discovery of this deletion supports localization of HTX1 to Xq26.2 and reveals the first molecular-genetic abnormality associated with human left-right-asymmetry defects.

Failure to detect connexin43 mutations in 38 cases of sporadic and familial heterotaxy.

BACKGROUND: Heterotaxy results from failure to establish normal left/right asymmetry during embryonic development. Typical manifestations include complex heart defects and malpositioning of abdominal organs. Missense base substitutions clustered in a 150-base pair region of the gap-junction gene connexin43 (cx43) have been implicated in the pathogenesis of heterotaxy. METHODS AND RESULTS: cx43 was studied in 38 cases of sporadic and familial heterotaxy. A 400-base pair region containing the previously reported mutation sites was amplified and directly sequenced in 19 patients. Nineteen additional patients were tested for restriction fragments predicted by two of the previously reported missense substitutions. No difference from normal control subjects was detected in any of the patients. CONCLUSIONS: Randomly selected cases of heterotaxy are unlikely to be the result of mutations in cx43.

Major histocompatibility complex class II molecule expression on muscle cells is regulated by differentiation: implications for the immunopathogenesis of muscle autoimmune diseases.

Major histocompatibility complex (MHC) class II molecules are expressed on myoblasts after interferon-gamma (IFN-gamma) treatment, suggesting a muscle cell involvement in antigen presentation in inflammatory myopathies. However, they were not observed on normal or pathological myofibers. This discrepancy might be related to different responsiveness of developmentally differentiated muscle cells to IFN-gamma. Myoblasts expressed class II transcripts and proteins after IFN-gamma, while myotubes and innervated contracting muscle cells did not show staining for class II molecules. At all cell stages no loss of IFN-gamma receptor was detected indicating that myofiber maturation blocks their capacity to express MHC class II molecules. This suggests that completely differentiated myofibers cannot participate in class II restricted immunological reactions.

Lack of mRNA and dystrophin expression in DMD patients three months after myoblast transfer.

We report our experience on myoblast transplantation in three Duchenne muscular dystrophy patients. Pure myoblasts (55 x 10(6) per patient) from HLA-matched donors, were injected into a tibialis anterior and the controlateral muscle was sham injected. Three months after transplantation, biopsies from the injected muscles were negative for dystrophin expression by immunocytochemistry. Reverse transcriptase-PCR (RT-PCR) failed to amplify any fragments of the deleted regions. This result confirms that myoblast transplantation is feasible, although the efficacy of this therapeutic approach is poor.

Cloning of the gene for ocular albinism type 1 from the distal short arm of the X chromosome.

Ocular albinism type 1 (OA1) is an X-linked disorder characterized by severe impairment of visual acuity, retinal hypopigmentation and the presence of macromelanosomes. We isolated a novel transcript from the OA1 critical region in Xp22.3-22.2 which is expressed at high levels in RNA samples from retina, including the retinal pigment epithelium, and from melanoma. This gene encodes a protein of 424 amino acids displaying several putative transmembrane domains and sharing no similarities with previously identified molecules. Five intragenic deletions and a 2 bp insertion resulting in a premature stop codon were identified from DNA analysis of patients with OA1, indicating that we have identified the OA1 gene.

A cluster of sulfatase genes on Xp22.3: mutations in chondrodysplasia punctata (CDPX) and implications for warfarin embryopathy.

X-linked recessive chondrodysplasia punctata (CDPX) is a congenital defect of bone and cartilage development characterized by aberrant bone mineralization, severe underdevelopment of nasal cartilage, and distal phalangeal hypoplasia. A virtually identical phenotype is observed in the warfarin embryopathy, which is due to the teratogenic effects of coumarin derivatives during pregnancy. We have cloned the genomic region within Xp22.3 where the CDPX gene has been assigned and isolated three adjacent genes showing highly significant homology to the sulfatase gene family. Point mutations in one of these genes were identified in five patients with CDPX. Expression of this gene in COS cells resulted in a heat-labile arylsulfatase activity that is inhibited by warfarin. A deficiency of a heat-labile arylsulfatase activity was demonstrated in patients with deletions spanning the CDPX region. These data indicate that CDPX is caused by an inherited deficiency of a novel sulfatase and suggest that warfarin embryopathy might involve drug-induced inhibition of the same enzyme.

Fetus-like dystrophin expression and other cytoskeletal protein abnormalities in centronuclear myopathies.

We have investigated supposed maturational arrest of muscle in centronuclear myopathies (CNMs) by characterizing the expression of dystrophin, other cytoskeletal proteins, and fetal myosin in the muscle fibers of 9 CNM patients (4 sporadic, 3 familial, 2 adult sporadic). Dystrophin and beta-spectrin localized intracytoplasmically in centrally nucleated fibers. Talin and vinculin were normally expressed. Desmin was radially organized in several fibers in all patients. Scattered vimentinpositive fibers were found in 3 cases. Six myotonic dystrophy cases and 4 inflammatory myopathy cases with regenerating fibers were also studied: dystrophin and the membrane cytoskeletal proteins were normally expressed in the former; and dystrophin, spectrin, and vinculin were reduced in the latter. Intracytoplasmic dystrophin is further evidence of maturational arrest in CNMs. Spectrin and dystrophin codistribute in these pathological conditions as in normal muscle. We conclude that the altered cytoskeletal network found in CNMs likely plays a pathogenetic role in these conditions.

Dystrophin abnormalities in Duchenne and Becker dystrophy carriers: correlation with cytoskeletal proteins and myosins.

Characterization with a panel of six antibodies revealed abnormal dystrophin expression in 6 of 20 Duchenne muscular dystrophy (DMD) carriers examined, and in 5 of 12 Becker muscular dystrophy (BMD) carriers examined. The immunocytochemistry of muscle fibres was normal with five of the antibodies in two BMD carriers, but some muscle fibres were negative to the antibody directed against a portion of the dystrophin rod domain. Mosaicism was detected with all six antibodies in the other three BMD (but in only a small number of fibres) and in all DMD carriers muscles. Spectrin, vinculin and talin were immunolocalized in the same muscle specimens in order to assess membrane cytoskeletal integrity and to correlate their expression with that of dystrophin. These proteins, including vinculin, which was previously reported to be reduced in DMD patient muscles, were normally present on the surface of all dystrophin-deficient fibres. Muscle fibre types were characterized using monoclonal antibodies against fetal myosin and adult fast and adult slow myosin heavy chains. In both the DMD and BMD carriers, a significant reduction in type 2B fibres, as well as an increase in type 2C and fetal myosin-containing fibres was found - as has also been reported in DMD patients. Altered dystrophin expression was observed more frequently in type 2 than type 1 fibres. Dystrophin deficiency was found in a high percentage of type 2C fibres as well as in all fibres expressing fetal myosin; this suggests that dystrophin-deficient fibres are more susceptible to degeneration, leading to regeneration.

Very small dystrophin molecule in a family with a mild form of Becker dystrophy.

The genetic defect in a family with a mild form of Becker dystrophy was characterized by immunocytochemical, immunoblot and genomic DNA analysis in two patients and a carrier. Immunocytochemical localization on muscle preparations with a series of antibodies against different regions of the dystrophin molecule showed normal dystrophin expression with all the antibodies except anti-30 kDa antiserum. In the carrier's muscle, mosaicism was observed only with the anti-30 kDa. Immunoblot analysis revealed a band of about 250 kDa in the patients' muscles and a double band of normal and of reduced weight protein in carrier muscle. In the patients Multiplex-PCR (M-PCR) and Southern blot revealed deletions from exon 13 to exon 41. The study confirms that very mild Becker muscular dystrophy can be associated with a large intragenic deletion from the dystrophin gene.

Interactions of manganese with human brain glutathione-S-transferase.

Chronic exposure to manganese-laden dusts induces, in humans and lower primates, neurological disorders with clinicopathological features that resemble idiopathic Parkinson's disease. As many authors have suggested, manganese neurotoxicity could be related to the capability of this metal to increase catechol autoxidation in catecholaminergic neurons, therefore increasing the formation of toxic compounds such as peroxides, superoxides, free radicals, and semi-orthoquinones. Oxidative stresses and consequent neuronal damage could then occur if physiological scavenger mechanisms fail in their detoxifying action. We here report that manganese chloride weakly inhibits, in a dose-dependent way by a reversible competitive mechanism, human brain glutathione-S-transferases possibly suggesting that manganese intoxication could cause intraneuronal accumulation of cytotoxic compounds. We also report that both 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, a neurotoxin known to induce in man Parkinson-like syndromes, and one of its metabolites 1-methyl-4-phenylpyridinium failed to decrease glutathione-S-transferase activity.