Developmental MYH3 Myopathy Associated with Expression of Mutant Protein and Reduced Expression Levels of Embryonic MyHC.

OBJECTIVE: An essential role for embryonic MyHC in foetal development has been found from its association with distal arthrogryposis syndromes, a heterogeneous group of disorders characterised by congenital contractions. The latter probably result from severe myopathy during foetal development. Lack of embryonic muscle biopsy material and suitable animal models has hindered study of the pathomechanisms linking mutations in MYH3 to prenatal myopathy. METHODS AND RESULTS: We determined the pathomechanisms of developmental myopathy caused by recurrent p.Thr178Ile MYH3 heterozygosity, using patient-derived skeletal muscle cells in culture as an experimental disease model to emulate early embryonic development. These cultured cells were processed for discrimination and quantitative analysis of mutant and wild-type MYH3 alleles and MyHC transcripts, real-time RT-qPCR, sequence analysis, immunofluorescence microscopy, immunoblot, and proteomic assessments. Involvement of the ubiquitin proteasome system was investigated in patients with p.Thr178Ile mutations in MYH3 and MYH2. We found equal overall expression of mutant and wild-type MyHC mRNAs and proteins. Compared to the controls, however, expression of embryonic MyHC transcripts and proteins was reduced whereas expression of myosin-specific E3 ubiquitin ligase (MuRF1) was increased. We also found delayed myofibrillogenesis and atrophic myotubes but structured sarcomeres. CONCLUSION: In conclusion, this study suggests that developmental p.Thr178Ile MYH3 myopathy is associated with a combined pathomechanism of insufficient dosage of functional embryonic MyHC and production of mutant protein.

[Genetic and prenatal diagnosis for a Chinese family with primary carnitine deficiency].

OBJECTIVE: To identify potential mutation of SLC22A5 gene in a 5-month-old boy affected with primary carnitine deficiency and provide genetic counseling and prenatal diagnosis for the members of his family. METHODS: DNA was extracted from peripheral blood samples derived from the proband, his parents and elder sister, as well as amniotic fluid from his pregnant mother. All of the 10 exons of the SLC22A5 gene were amplified by PCR and subjected to Sanger sequencing. The amniotic fluid sample was also subjected to G-banded karyotyping and multiplex ligation-dependent probe amplification (MLPA). RESULTS: A homozygous mutation c.760C>T (p.R254X) of the SLC22A5 gene was detected in the proband. Heterozygous mutation c.760C>T (p.R254X) was also found in other family members including the fetus. The karyotyping and chromosomal microdeletion testing for the amniotic fluid sample were both normal. CONCLUSION: The newly identified homozygous nonsense c.760C>T (p.R254X) mutation of the SLC22A5 gene probably underlies the primary carnitine deficiency of the proband. Genetic counseling and prenatal diagnosis have been provided for this family.

Aetiology and pathogenesis of IUGR.

Intrauterine growth restriction (IUGR) is a major cause of perinatal mortality and morbidity. A complex and dynamic interaction of maternal, placental and fetal environment is involved in ensuring normal fetal growth. An imbalance or lack of coordination in this complex system may lead to IUGR. Animal studies have given us an insight into some aspects of the basic pathophysiology of IUGR, and recent technologies such as Doppler studies of maternal and fetal vessels have added further information. The aetiologies of IUGR are diverse, involving multiple complex mechanisms, which make understanding of the pathophysiology difficult. However, particular focus is placed on the mechanisms involved in uteroplacental insufficiency as a cause of IUGR, as (1) it is common, (2) outcome can be good if timing of delivery is optimal and (3) it may be amenable to therapy in the future. While the research into the pathophysiology of IUGR continues, there have been interesting discoveries related to the genetic contribution to IUGR and the intrauterine programming of adult-onset diseases attributed to IUGR.

Myoblasts isolated from hypertrophy-responsive callipyge muscles show altered growth rates and increased resistance to serum deprivation-induced apoptosis.

Back and hind limb muscles of sheep paternally heterozygous for the callipyge single nucleotide polymorphism undergo extensive hypertrophy shortly after birth. We have established cell cultures from foetal semitendinosus and longissimus dorsi muscles of normal and callipyge animals. Cultures were assessed for rates of proliferation, cell death, myogenicity and DLK1 expression. Myoblasts from callipyge semitendinosus, but not longissimus dorsi muscles, proliferated faster than myoblasts isolated from normal semitendinosus muscle, and cells isolated from either callipyge muscle were more resistant to serum deprivation-induced apoptosis than equivalent cells isolated from normal individuals. These observations indicate that there are intrinsic differences in the behaviour of isolated myoblasts, which are associated with their muscle and genotype of origin. As myoblasts are the cells responsible for hypertrophy of muscle fibres, the observed differences in cell growth may play a role in the hypertrophy of certain muscles in callipyge animals.

Prenatal diagnosis of myopathy, encephalopathy, lactic acidosis, and stroke-like syndrome: contribution to understanding mitochondrial DNA segregation during human embryofetal development.

INTRODUCTION: Myopathy, encephalopathy, lactic acidosis, and stroke-like (MELAS) syndrome, a maternally inherited disorder that is among the most common mitochondrial DNA (mtDNA) diseases, is usually associated with the m.3242A>G mutation of the mitochondrial tRNA(leu) gene. Very few data are available with respect to prenatal diagnosis of this serious disease. The rate of mutant versus wild-type mtDNA (heteroplasmy) in fetal DNA is indeed considered to be a poor indicator of postnatal outcome. MATERIALS AND METHODS: Taking advantage of a novel semi-quantitative polymerase chain reaction test for m.3243A>G mutant load assessment, we carried out nine prenatal diagnoses in five unrelated women, using two different fetal tissues (chorionic villi v amniocytes) sampled at two or three different stages of pregnancy. RESULTS: Two of the five women, although not carrying m.3243A>G in blood or extra-blood tissues, were, however, considered at risk for transmission of the mutation, as they were closely related to MELAS-affected individuals. The absence of 3243A>G in the blood of first degree relatives was associated with no mutated mtDNA in the cardiovascular system (CVS) or amniocytes, and their three children are healthy, with a follow-up of 3 months-3 years. Among the six fetuses from the three carrier women, three were shown to be homoplasmic (0% mutant load), the remaining three being heteroplasmic, with a mutant load ranging from 23% to 63%. The fetal mutant load was fairly stable at two or three different stages of pregnancy in CVS and amniocytes. Although pregnancy was terminated in the case of the fetus with a 63% mutant load, all other children are healthy with a follow-up of 3 months-6 years. CONCLUSION: These data suggest that a prenatal diagnosis for MELAS syndrome might be helpful for at-risk families.

[Progress in pediatric neurology]. / Progrès en neurologie pédiatrique.

A central part of Pediatric Neurology is currently dominated by the search for genetic factors involved in developmental disorders of the nervous system, including cases where the cytogenetic examination remains uncontributive. The prerequisite for a good definition of the malformative phenotypes leads to distinguish: 1 cerebral malformations that can be identified at the macroscopic scale, by imaging. 2 polymalformative syndromes including mental retardation where cerebral imaging is not contributive, thus the syndromatic definition is based on associated somatic anomalies. 3 Non-syndromatic mental retardation, where a genetic origin is clear only in the familial forms. Various methodological approaches have included genetic linkage studies, search for inframicroscopic chromosomal rearrangements in the critical region and investigation of candidate genes. A great number of syndromes have been connected with a great diversity of genetic mechanisms, whose many examples are presented: genopathies with regular or variable expression, unstable mutations, contiguous gene syndromes or other complex infracytogenetic rearrangements, chromosomal or genic mosaicisms, mutations submitted to parental imprinting. New methods of genomic screening will be necessary to progress in this field, given the great number of genes involved in cerebral development. As for the early developmental disorders of the PNS and muscle, their diagnosis becomes frequent during the intrauterine life, raising the problem of a better definition of the fetopathological phenotypes.

New insights into the pathogenesis of congenital myopathies.

Congenital myopathies are developmental disorders of muscle that are best understood in the context of ontogenesis. Segmental amyoplasia results from a defective somite, usually because of lack of induction by the notochord and neural tube; the connective tissue matrix of the muscle is derived from lateral mesoderm and is present, but the myocytes are derived from somitic mesoderm and are replaced by adipose cells. Generalized amyoplasia is due to defective myogenic regulatory genes. X-linked recessive myotubular myopathy is associated with overexpression of vimentin and desmin, fetal intermediate filaments that attach to nuclear, mitochondrial, and inner sarcolemmal membranes and Z-bands of sarcomeres to preserve the morphologic organization of the myotube. Neonatal myotonic dystrophy is a true maturational delay in muscle development. Congenital muscle fiber-type disproportion is a syndrome of multiple etiologies but in some cases is associated with cerebellar hypoplasia and may be the result of abnormal suprasegmental stimulation of the developing motor unit at 20 to 28 weeks' gestation, mediated through bulbospinal pathways but not the corticospinal tract. Maturational delay of muscle in late developmental stages is less specific than in stages before midgestation. The Proteus syndrome is a muscular dysgenesis; abnormal paracrine growth factors and perhaps altered genes that regulate muscle differentiation and growth, such as myoD and myogenin, are the suspected cause. Focal proliferative myositis may be another example of a "paracrine myopathy."

Delayed somite formation in a quail line exhibiting myofiber hyperplasia is accompanied by delayed expression of myogenic regulatory factors and myosin heavy chain.

A myofiber hyperplastic quail line P has been developed through selection for heavy body weight. Since the number of muscle fibers is determined early in development and skeletal muscle originates from somites, we compared somite formation and muscle-specific gene expression in P- and control C-line quail embryos. At 47 hours of incubation, C embryos had 18 somite pairs and P embryos had 14.3. By 72 and 120 hours, both lines appeared to be at the same stage of somite development. To determine whether the delay in the formation of the brachial somites was accompanied by alterations in muscle-specific gene expression, we conducted whole-mount in situ hybridization and immunofluorescence studies. At 47 hours of incubation, C embryos were expressing qmf1 in the first 12 somites, while in P embryos only the first 7 somites showed qmf1 activation. Delays in expression were also observed for qmf3 at 43 hours and for all three myogenic factors (qmf1, qmf2 and qmf3) at 60 hours. At 65 hours, C embryos expressed myosin heavy chain in the first 15 somite pairs and P embryos in the first 7. At 72 hours, the transient delay in somite formation had disappeared and there was no lag in myosin heavy chain expression between the lines. The phase delay in brachial somite formation, myogenic factors and myosin heavy chain expression may be associated with the observed myofiber hyperplasia in P-line quail by allowing an increase in the muscle stem cell population.

Barium currents in developing skeletal muscle cells of normal and mutant mice foetuses with 'muscular dysgenesis'.

The ontogenesis of Ca channel activities was studied in the developing myotubes of normal mice and mutant mice foetuses with 'Muscular Dysgenesis'. The ionic current through Ca channels was measured with Ba2+ as charge carrier using the whole cell clamp technique. All dissociated myotubes from foetuses (14th to 18th day of gestation) showed two distinct inward Ba currents: a low threshold, transient current (T-type) and a high threshold sustained current. In normal myotubes, T-type current density increased from the 14th day to the 16th day of gestation. After day 16, T-type current density decreased gradually until birth. Similar changes in T-type current density were observed in developing dysgenic myotubes where the current density was about 40% of that measured in normal myotubes throughout the prenatal period studied. The high threshold sustained current (L-type current) density increased gradually with age in normal myotubes while absent in dysgenic muscle. The latter, regardless of age, showed a high threshold current (Idys) which is distinct from the L-type current. Idys density did not change during the prenatal myogenesis period studied.

Pathogenetic mechanisms of fetal akinesia deformation sequence and oligohydramnios sequence.

This article briefly reviews the participation of fetal compression, muscular weakness, and fetal akinesia in the genesis of the anomalies found in fetal akinesia deformation sequence (FADS) and oligohydramnios sequence (OS). Both sequences share phenotypic manifestations, such as arthrogryposis, short umbilical cord, and lung hypoplasia, in relation to decreased intrauterine fetal motility. Other characteristic manifestations found in OS, such as Potter face, and redundant skin, are produced by fetal compression. On the other hand, growth retardation, craniofacial anomalies, micrognathia, long bone hypoplasia, and polyhydramnios found in FADS could be related to intrauterine muscular weakness.

Myotubular myopathy: arrest of morphogenesis of myofibres associated with persistence of fetal vimentin and desmin. Four cases compared with fetal and neonatal muscle.

Vastus lateralis muscle biopsies of four unrelated male neonates showing myotubular (i.e. centronuclear) myopathy (MM) were compared with muscle from four human fetuses in the myotubular stage of development, a 31 week preterm infant and four term neonates. The perimysium, blood vessels, spindles, myelinated intramuscular nerves, and motor end-plates in MM are as well developed as in term neonatal muscle. The cytoarchitecture of myofibres in MM is more mature than that of fetal myotubes in the spacing of central nuclei, Z-band registry, development of the sarcotubular system, and in the condensation of nuclear chromatin and nucleoli. Triads in MM may retain an immature oblique or longitudinal orientation. Myofibrillar ATPase shows normal differentiation of fibre types, consistent with normal innervation. Spinal motor neurons are normal in number and in RNA fluorescence. Immunoreactivity for vimentin and desmin in myofibres of MM is uniformly strong, as in fetal myotubes and unlike mature neonatal muscle. Maternal muscle biopsies of two cases also showed scattered small centronuclear myofibres reactive for vimentin and desmin. The arrest in morphogenesis of fibre architecture in MM is not a general arrest in muscle development. Persistence of fetal cytoskeletal proteins that preserve the immature central positions of nuclei and mitochondria may be important in pathogenesis. Vimentin/desmin studies of the infant and maternal muscle biopsies are useful in establishing the diagnosis.

Muscular dysgenesis in fowl: ultrastructural study of skeletal muscles in the crooked neck dwarf (cn/cn) mutant.

In vivo evolutive aspects of muscular dysgenesis were studied in normal and crooked neck dwarf (cn/cn) 7.5- to 20-day chick embryos. Wing, leg and breast muscles were processed for electron microscopy. It appears that the effects of the gene cn are expressed in the multinucleated cells as fine structural aberrations. Dilatation of the sarcotubular system, partial loss of the contractile elements and malorganization of the myofibrils are the major anomalies observed from day 7.5 to 18 of incubation. These changes do not constitute an abrupt phenomenon. Normal and diseased multinucleated cells always coexist in the same muscle specimen; however the frequency of the pathological cells augments with time. At the end of the incubation period, the poorly organized muscle tissue contains only morbid muscle cells. Phagocytosis or autolysis are absent.

Abnormal transverse tubule system and abnormal amount of receptors for Ca2+ channel inhibitors of the dihydropyridine family in skeletal muscle from mice with embryonic muscular dysgenesis.

We have found two important sets of abnormalities in skeletal muscle from mice with embryonic muscular dysgenesis. These abnormalities involve the internal structural organization of the muscle fiber and its content of voltage-dependent Ca2+ channels. The first abnormality concerns the ultrastructural aspects of the membranous couplings between sarcoplasmic reticulum and the transverse tubules, known as triads. The triads are less numerous, are disorganized, and lack spaced densities (feet). The second abnormality is a significant decrease in specific binding sites for the dihydropyridine derivatives, (known as Ca2+ channel inhibitors) in striated skeletal muscle, but not in cardiac muscle. Both sets of abnormalities are potentially directly linked to the uncoupling of excitation and contraction.

Is central core disease with structural core a fetal defect?

Morphological and biochemical studies were performed in three cases of congenital non-progressive myopathy in two generations of the same family. In the muscle biopsy nearly all the fibres were uniform in enzyme activity and belonged to type 2 C. Typical structural central cores were observed in 90% of the muscle fibres. Some ultrastructural characteristics of the core area, as well as disturbances of the myofibrillar proteins pattern, seen in the examined cases suggest that core formation may be a result of protein synthesis disturbances in an early stage of myogenesis.

The arthrogrypotic foot plan of management and results of treatment.

Fifty-one arthrogrypotic feet have been treated and followed by the Pediatric Orthopaedic Unit, Tufts New England Medical Center, (1970-1980). Forty of the 51 feet presented as equinovarus with the residual divided among metatarsus adductus, vertical tali, and calcaneovalgus. Equinovarus deformities are the most resistant in all cases. Corrective casts are applied for at least the first 3 months of life. Surgical procedures were then initiated with any evidence of lack of progression of treatment. Varus and equinus were treated by an extensive posterior and medial release. Lateral soft tissue releases in addition to calcaneocuboid fusion or cuboid osteotomy were necessary in 24 of the 70 operations. Recurrence rate has been a problem in the simple type of posterior release including only an Achilles tendon lengthening, and posterior capsulotomy of the ankle and subtalar joint. Talectomy has been carried out in four feet and appears to be one type of reasonable salvage procedure in smaller children with recurrent varus. Treatment is difficult in these patients but a plantigrade foot should be achieved in all cases.

Extensive multiple innervation and abnormal synaptogenesis in muscular dysgenesis (mdg/mdg) in the mouse embryo.

Muscular dysgenesis (mdg) is an autosomal recessive mutation in the mouse characterized by total muscle inactivity in vivo or in vitro. The muscle fiber in the mdg/mdg diaphragm was not only morphologically abnormal but also multiply innervated; the motor innervation was very dense, showing overgrowth and sprouting. As expected at the ultrastructural level, nerve-muscle contacts were composed of dense appositions of numerous axon terminals (dense focal polyinnervation). Moreover, these mdg/mdg neuromuscular junctions, lacking post-synaptic unfolding, were immature compared to the control ones. This retarded neuromuscular junction differentiation in muscular dysgenesis may be related to considerable delay in muscle maturation and/or abnormal muscular differentiation, or to a nerve defect independent of, or causally related to, the muscular defect.

Histological study of muscular hypoplasia in the crooked neck dwarf mutant (cn/cn) chick embryo.

Tibiotarsal segments of 12-day chick embryos homozygous for the crooked neck dwarf gene (cn/cn) were examined histologically following routine methods of preparation. The myogenic mass fails to divide into separate muscle bundles during the early stages of differentiation. Myoblasts and myotubes are observed, although the proportion favors the mononucleate cell population. Multinucleate myotubes are often wavy in appearance and many contain eosinophilic cytoplasmic inclusions. The entire tissue mass of mutants appears more compacted than in control limbs. Poor organization of muscle appears related to the lack of a suitable connective tissue system. Epimysia, perimysia, and subcutaneous connective tissue fail to develop properly. Tendons are poorly developed or absent. Comparisons between mutant and control embryos show no differences in peripheral innervation. Nerve fascicles penetrate deeply into the developing muscle of both species. The distribution of vascular elements is seemingly normal also. Skeletal muscle of cn/cn embryos is capable of differentiating to the myotube stage, after which it undergoes cellular degeneration without achieving a functional state. Comparisons of this mutant with alleged chemical phenocopies show important differences.