Delayed cerebral edema and fatal coma after minor head trauma: role of the CACNA1A calcium channel subunit gene and relationship with familial hemiplegic migraine.

Trivial head trauma may be complicated by severe, sometimes even fatal, cerebral edema and coma occurring after a lucid interval ("delayed cerebral edema"). Attacks of familial hemiplegic migraine (FHM) can be triggered by minor head trauma and are sometimes accompanied by coma. Mutations in the CACNA1A calcium channel subunit gene on chromosome 19 are associated with a wide spectrum of mutation-specific episodic and chronic neurological disorders, including FHM with or without coma. We investigated the role of the CACNA1A gene in three subjects with delayed cerebral edema. Two subjects originated from a family with extreme FHM, and one subject was the previously asymptomatic daughter of a sporadic patient with hemiplegic migraine attacks. In all three subjects with delayed severe edema, we found a C-to-T substitution resulting in the substitution of serine for lysine at codon 218 (S218L) in the CACNA1A gene. The mutation was absent in nonaffected family members and 152 control individuals. Haplotype analysis excluded a common founder for both families. Neuropathological examination in one subject showed Purkinje cell loss with relative preservation of granule cells and sparing of the dentate and inferior olivary nuclei. We conclude that the novel S218L mutation in the CACNA1A calcium channel subunit gene is involved in FHM and delayed fatal cerebral edema and coma after minor head trauma. This finding may have important implications for the understanding and treatment of this dramatic syndrome.

Facioscapulohumeral muscular dystrophy.

A decade's progress in facioscapulohumeral muscular dystrophy genetics has been marked by the discovery of novel genetic phenomena such as crossover of subtelomeric DNA between chromosomes 4 and 10 in normal individuals and by the recognition that the facioscapulohumeral muscular dystrophy deletion-mutation may cause a position variegation effect on more proximal DNA. The mutated DNA itself is probably not transcribed. Larger deletions tend to cause more severe disease. Antenatal diagnosis, based on detection of the short fragment of mutated DNA, is possible in between 95 and 100% of cases, depending on the precise nature of the parental facioscapulohumeral muscular dystrophy mutation. Yet remarkably, the nature of the gene product(s) of the affected proximal gene(s), as well as of the molecular pathogenesis of facioscapulohumeral muscular dystrophy muscle, retinal and cochlear disease, is completely unknown. Marked perivascular inflammation is often present in facioscapulohumeral muscular dystrophy muscle biopsies. The expression of facioscapulohumeral muscular dystrophy within reported monozygotic twinships differs greatly. This raises the question of whether variations in expression of the T-cell receptor gene repertoire or of other immune genes play an important modifying role in determining the severity of facioscapulohumeral muscular dystrophy. A focus on traditional scientific disciplines may now be appropriate. Symptomatic treatments, for instance of scapular winging and of lagophthalmos, are important, and timely photocoagulation of the retinal exudates which are a very rare, but real, complication of retinal telangiectasis can curtail visual loss. The results of collobarative trials of pharmacological agents such as albuterol which affect muscle mass and development are awaited.

Myosin heavy chain composition of single fibres and their origins and distribution in developing fascicles of sheep tibialis cranialis muscles.

The myosin heavy chain (MHC) composition of single muscle fibres in developing sheep tibialis cranialis muscles was examined immunohistochemically with monoclonal antibodies to MHC isozymes. Data were collected with conventional microscopy and computerized image analysis from embryonic day (E) 76 to postnatal day (PN) 20, and from adult animals. At E76, 23% of the young myofibres stained for slow-twitch MHC. The number of these fibres considerably exceeded the number of primary and secondary myotubes. By E100, smaller fibres, negative for slow-twitch MHC, encircled each fibre from the initial population to form rosettes. A second population of small fibres appeared in the unoccupied spaces between rosettes. Small fibres, whether belonging to rosettes or not, did not initially express slow-twitch MHC, expressing mainly neonatal myosin instead. These small fibres then diverged into three separate groups. In the first group most fibres transiently expressed adult fast myosin (maximal at E110-E120), but in the adult expressed slow myosin. This transformation to the slow MHC phenotype commenced at E110, was nearing completion by 20 postnatal days, and was responsible for approximately 60% of the adult slow twitch fibre population. In the other two groups expression of adult fast MHC was maintained, and in the adult they accounted for 14% (IIa MHC) and 17% (IIb MHC) of the total fibre numbers. We conclude that muscle fibre formation in this large muscle involves at least three generations of myotube. Secondary myotubes are generated on a framework of primary myotubes and both populations differentiate into the young myofibres which we observed at E76 to form rosettes. Tertiary myotubes, in turn, appear in the spaces between rosettes and along the borders of fascicles, using the outer fibres of rosettes as scaffolds.

Neural control of the sequence of expression of myosin heavy chain isoforms in foetal mammalian muscles.

The expression of myosin isoforms was studied during development of calf muscles in foetal and neonatal rats, using monoclonal antibodies against slow, embryonic and neonatal isoforms of myosin heavy chain (MHC). Primary myotubes had appeared in all prospective rat calf muscles by embryonic day 16 (E16). On both E16 and E17, primary myotubes in all muscles with the exception of soleus stained for slow, embryonic and neonatal MHC isoforms; soleus did not express neonatal MHC. In earlier stages of muscle formation staining for the neonatal isoform was absent or faint. Secondary myotubes were present in all muscles by E18, and these stained for both embryonic and neonatal MHCs, but not slow. In mixed muscles, primary myotubes destined to differentiate into fast muscle fibres began to lose expression of slow MHC, and primary myotubes destined to become slow muscle fibres began to lose expression of neonatal MHC. This pattern was further accentuated by E19, when many primary myotubes stained for only one of these two isoforms. Chronic paralysis or denervation from E15 or earlier did not disrupt the normal sequence of maturation of primary myotubes up until E18, but secondary myotubes did not form. By E19, however, most primary myotubes in aneural or paralyzed tibialis anterior muscles had lost expression of slow MHC and expressed only embryonic and neonatal MHCs. Similar changes occurred in other muscles, except for soleus which never expressed neonatal MHC, as in controls. Paralysis or denervation commencing later than E15 did not have these effects, even though it was initiated well before the period of change in expression of MHC isoforms. In this case, some secondary myotubes appeared in treated muscles. Paralysis initiated on E15, followed by recovery 2 days later so that animals were motile during the period of change in expression of MHC isoforms, was as effective as full paralysis. These experiments define a critical period (E15-17) during which foetuses must be active if slow muscle fibres are to differentiate during E19-20. We suggest that changes in expression of MHC isoforms in primary myotubes depend on different populations of myoblasts fusing with the myotubes, and that the normal sequence of appearance of these myoblasts has a stage-dependent reliance on active innervation of foetal muscles. A critical period of nerve-dependence for these myoblasts occurs several days before their action can be noted.

Myonuclear birthdates distinguish the origins of primary and secondary myotubes in embryonic mammalian skeletal muscles.

Myotubes were isolated from enzymically disaggregated embryonic muscles and examined with light microscopy. Primary myotubes were seen as classic myotubes with chains of central nuclei within a tube of myofilaments, whereas secondary myotubes had a smaller diameter and more widely spaced nuclei. Primary myotubes could also be distinguished from secondary myotubes by their specific reaction with two monoclonal antibodies (MAbs) against adult slow myosin heavy chain (MHC). Myonuclei were birth dated with [3H]thymidine autoradiography or with 2-bromo-5'-deoxyuridine (BrdU) detected with a commercial monoclonal antibody. After a single pulse of label during the 1-2 day period when primary myotubes were forming, some primary myotubes had many myonuclei labelled, usually in adjacent groups, while in others no nuclei were labelled. If a pulse of label was administered after this time labelled myonuclei appeared in most secondary myotubes, while primary myotubes received few new nuclei. Labelled and unlabelled myonuclei were not grouped in the secondary myotubes, but were randomly interspersed. We conclude that primary myotubes form by a nearly synchronous fusion of myoblasts with similar birthdates. In contrast, secondary myotubes form in a progressive fashion, myoblasts with asynchronous birthdates fusing laterally with secondary myotubes at random positions along their length. These later-differentiating myoblasts do not fuse with primary myotubes, despite being closely apposed to their surface. Furthermore, they do not generally fuse with each other, as secondary myotube formation is initiated only in the region of the primary myotube endplate.

Immunocytochemical and electrophoretic analyses of changes in myosin gene expression in cat posterior temporalis muscle during postnatal development.

Changes in myosin gene expression during the postnatal development of the homogeneously superfast kitten posterior temporalis muscle were examined using immunocytochemical techniques supplemented by pyrophosphate gel electrophoresis and gel electrophoresis-derived enzyme linked immunosorbent assay (GEDELISA) of myosin isoforms. The antibodies used were polyclonals directed against the heavy chains of superfast and foetal myosins and monoclonals against the heavy chains of slow and fast myosins. The fibres of the posterior temporalis in the newborn kitten stained almost uniformly with the anti-foetal myosin antibody and the largest of these fibres stained strongly for superfast myosin. A subpopulation of fibres staining for superfast myosin also stained lightly for slow myosin. These slow staining fibres were evenly distributed in the centres of muscle fibre bundles, reminiscent of primary fibres in limb fast muscle. During subsequent development, slow myosin staining disappeared and superfast myosin replaced foetal myosin so that by 50 days the muscle was virtually homogeneously superfast as in the adult. Fast myosin was never expressed at any stage. It is proposed that fibres staining transiently for slow myosin are superfast primary fibres which are homologous to fast primary fibres recently described in regions of limb muscles devoid of slow fibres in the matured animal. Other jaw-closing muscles have significant populations of slow fibres in the mature animal and it is postulated that there exists in these muscles a second class of jaw primary fibres, the slow primary fibres, in which slow myosin synthesis would be sustained in the adult. It is suggested that the myogenic cells of jaw-closing and limb muscles are of two distinct types preprogrammed to express different muscle genes.

Immunocytochemical and electrophoretic analyses of changes in myosin gene expression in cat limb fast and slow muscles during postnatal development.

Changes in myosin synthesis during the postnatal development of the fast extensor digitorum longus (EDL) and the slow soleus muscles of the kitten were examined using immunocytochemical techniques supplemented by pyrophosphate gel electrophoresis and gel electrophoresis-derived enzyme linked immunosorbent assay (GEDELISA) of myosin isoforms. The antibodies used were monoclonals against heavy chains of slow and fast myosins and a polyclonal against foetal/embryonic myosin. In both muscles in the newborn kitten, there was a population of more mature fibres which stained strongly for slow but weakly for foetal/embryonic myosin. These fibres were considered to be primary fibres. They formed 4.8% of EDL fibres and 26% of soleus fibres at birth, and continued to express slow myosin in adult muscles. The less mature secondary fibres stained strongly for foetal/embryonic myosin, and these could be divided into two subpopulations; fast secondaries in which foetal/embryonic myosin was replaced by fast myosin, and slow secondaries in which the myosin was replaced by slow myosin. At 50 days the EDL had a large population of fast secondaries (83% of total fibres) and a small population of slow secondaries which gradually transformed into fast fibres with maturity. The vast majority of secondary fibres in the soleus were slow secondaries, in which slow myosin synthesis persisted in adult life. There was a restricted zone of fast secondaries in the soleus, and these gradually transformed into slow fibres in adult life. It is proposed that the emergence of primary fibres and the two populations of secondary fibres is myogenically determined.

Ultrastructural identification of type 1 fibres in human skeletal muscle. Immunogold labelling of thin cryosections with a monoclonal antibody against slow myosin.

Existing methods for the ultrastructural identification of fibre types in human skeletal muscle are fallible. This has prompted us to develop a reliable immunoelectron microscopic approach for the identification of human skeletal muscle fibre types. Here we report the unambiguous electron microscopic identification of human type 1 muscle fibres, achieved by combining cryoultramicrotomy with colloidal gold immunocytochemical labelling, using a monoclonal antibody (N0Q7.5.4D) which is specific for the heavy chain of the slow myosin isoform of human skeletal muscle. This method for the identification of muscle fibre types and determination of myosin isoform distributions may have important applications in the ultrastructural study of pathological muscle and in the analysis of myofibrillar assembly during myogenesis.

Primary, secondary and tertiary myotubes in developing skeletal muscle: a new approach to the analysis of human myogenesis.

Monoclonal antibodies to myosins have been used to describe and define the appearance and maturation of 3 different classes of myotube in developing human quadriceps muscle. Five monoclonal antibodies were used: (i) MAb A against human slow myosin heavy chain; (ii) MAb B against a myosin heavy chain present in most adult Type 2 fibres; (iii) MAb C against myosin heavy chain present in all mature and immature Type 2 fibres; (iv) MAb D, with similar reactivity to MAb C; (v) MAb E against human embryonic myosin. The combined use of two of these antibodies (A and B) enables the confident early identification of each of 3 classes (primary, secondary, tertiary) of myotubes, which appear sequentially during myogenesis. Our results show that induction of slow myosin heavy chain synthesis is a biphasic phenomenon in developing human skeletal muscle. Slow myosin heavy chain was present in all the earliest (9 weeks gestation) primary myotubes, but was not detected in secondary or tertiary myotubes until about 29 weeks gestation. Each stage of fetal muscle development has a characteristic immunocytochemical pattern which reveals cellular heterogeneity not evident on myosin ATPase histochemistry. Myosin immunocytochemistry may usefully be applied to assess the gestational age of fetuses. A new interpretation of human skeletal muscle development is proposed, based on the separate programming of 3 different kinds of myotube. This may be important in the analysis of diseased muscle in which developmental abnormalities or regeneration are present.

Retinal vascular abnormalities in facioscapulohumeral muscular dystrophy. A general association with genetic and therapeutic implications.

Because of occasional reports of exudative retinal detachment with facioscapulohumeral muscular dystrophy (FSH) and deafness, we sought to determine by fluorescein angiography whether there is any general relationship between FSH muscular dystrophy and retinal vascular disease. Peripheral retinal capillary abnormalities, comprising telangiectasis, closure, leakage and microaneurysm formation, were demonstrated by angiography in 56 out of 75 individuals with clinical or genetic evidence of FSH. Only 3 patients had relevant ophthalmoscopic abnormalities of the posterior pole and in only 1 was there consequent visual loss. This study included one FSH family in which the propositus was treated for exudative retinopathy and 13 other subjects had telangiectasis, and 8 cases (including 3 parents of apparently 'sporadic' FSH cases) in which fluorescein angiography confirmed the abnormal genotype, even though clinical examination of skeletal muscle revealed no clear abnormality. There was no correlation between the severity of the muscle disease and the extent of the retinal vascular abnormality. Visual complications of telangiectasis, although rare, may present early in life and before there is overt evidence of muscle disease. Since visual loss may be preventable, ophthalmic examination should be undertaken on infants at risk of having the abnormal gene. The findings support the hypothesis that retinal capillary abnormalities are an integral part of the FSH muscular dystrophy syndrome and raise the question as to whether analogous capillary abnormalities could be implicated in the pathogenesis of FSH muscle disease.

Slow myosin in developing rat skeletal muscle.

Through S1 nuclease mapping using a specific cDNA probe, we demonstrate that the slow myosin heavy-chain (MHC) gene, characteristic of adult soleus, is expressed in bulk hind limb muscle obtained from the 18-d rat fetus. We support these results by use of a monoclonal antibody (mAb) which is highly specific to the adult slow MHC. Immunoblots of MHC peptide maps show the same peptides, uniquely recognized by this antibody in adult soleus, are also identified in 18-d fetal limb muscle. Thus synthesis of slow myosin is an early event in skeletal myogenesis and is expressed concurrently with embryonic myosin. By immunofluorescence we demonstrate that in the 16-d fetus all primary myotubes in future fast and future slow muscles homogeneously express slow as well as embryonic myosin. Fiber heterogeneity arises owing to a developmentally regulated inhibition of slow MHC accumulation as muscles are progressively assembled from successive orders of cells. Assembly involves addition of new, superficial areas of the anterior tibial muscle (AT) and extensor digitorum longus muscle (EDL) in which primary cells initially stain weakly or are unstained with the slow mAb. In the developing AT and EDL, expression of slow myosin is unstable and is progressively restricted as these muscles specialize more and more towards the fast phenotype. Slow fibers persisting in deep portions of the adult EDL and AT are interpreted as vestiges of the original muscle primordium. A comparable inhibition of slow MHC accumulation occurs in the developing soleus but involves secondary, not primary, cells. Our results show that the fate of secondary cells is flexible and is spatially determined. By RIA we show that the relative proportions of slow MHC are fivefold greater in the soleus than in the EDL or AT at birth. After neonatal denervation, concentrations of slow MHC in the soleus rapidly decline, and we hypothesize that, in this muscle, the nerve protects and amplifies initial programs of slow MHC synthesis. Conversely, the content of slow MHC rises in the neonatally denervated EDL. This suggests that as the nerve amplifies fast MHC accumulation in the developing EDL, accumulation of slow MHC is inhibited in an antithetic fashion. Studies with phenylthiouracil-induced hypothyroidism indicate that inhibition of slow MHC accumulation in the EDL and AT is not initially under thyroid regulation. At later stages, the development of thyroid function plays a role in inhibiting slow MHC accumulation in the differentiating EDL and AT.(ABSTRACT TRUNCATED AT 400 WORDS)

Retinal telangiectasis in facioscapulohumeral muscular dystrophy with deafness.

A 22-year-old patient with newly diagnosed facioscapulohumeral (FSH) muscular dystrophy had a macular lesion in her right eye and poor central vision, which had been present since early childhood. Fluorescein angiographic examination revealed bilateral peripheral vessel closure, peripheral retinal telangiectasis, and hyperfluorescence in both foveae. This widespread vascular abnormality was deemed responsible for her macular disease. Her mother, brother, and sister, all of whom are affected by varying degrees of FSH muscular dystrophy and clinical deafness, also have abnormal retinal vasculature, as determined by fluorescein angiography. However, none had related visual symptoms and two showed no ophthalmoscopic evidence of vascular abnormalities. In young patients with unexplained retinal vascular lesions, the diagnosis of FSH muscular dystrophy should be considered. Similarly, young patients with FSH muscular dystrophy should be examined for sight-threatening and potentially treatable vascular retinopathy.

Migraine coma. Meningitic migraine with cerebral oedema associated with a new form of autosomal dominant cerebellar ataxia.

A family with hemiplegic migraine has been documented for a period of over forty years. From this study and the literature we conclude that (1) migraine is a cause of recurrent coma which may be associated with life-threatening cerebral hemisphere oedema; (2) hyperpyrexia with CSF pleocytosis occurs in hemiplegic migraine, which may thus simulate viral meningoencephalitis; and (3) cerebral angiography is hazardous in hemiplegic migraine and may exacerbate coma and cerebral oedema. In the family reported, cerebellar ataxia was present during recovery from attacks of hemiplegic migraine and affected patients ultimately suffered from persistent ataxia with radiological cerebellar atrophy. This syndrome thus constitutes a distinct form of late-onset autosomal dominant cerebellar ataxia and also of familial periodic ataxia. The status of 'cerebellar migraine' is reviewed.

Kidney length in the newborn measured by ultrasound.

Kidney length was measured in 115 unselected neonates with portable real-time ultrasound. The left kidney was slightly, but significantly, longer than the right. There was a positive correlation between kidney length and birthweight, but kidney size did not increase significantly after 36 weeks gestation. Further ultrasound studies of the newborn kidney will be of interest.