Christoph Hübner

Mutations in the gene encoding immunoglobulin μ-binding protein 2 cause spinal muscular atrophy with respiratory distress type 1

Classic spinal muscular atrophy (SMA) is caused by mutations in the telomeric copy of SMN1. Its product is involved in various cellular processes, including cytoplasmic assembly of spliceosomal small nuclear ribonucleoproteins, pre-mRNA processing and activation of transcription. Spinal muscular atrophy with respiratory distress (SMARD) is clinically and genetically distinct from SMA. Here we demonstrate that SMARD type 1 (SMARD1) results from mutations in the gene encoding immunoglobulin μ-binding protein 2 (IGHMBP2; on chromosome 11q13.2–q13.4). In six SMARD1 families, we detected three recessive missense mutations (exons 5, 11 and 12), two nonsense mutations (exons 2 and 5), one frameshift deletion (exon 5) and one splice donor-site mutation (intron 13). Mutations in mouse Ighmbp2 (ref. 14) have been shown to be responsible for spinal muscular atrophy in the neuromuscular degeneration (nmd) mouse, whose phenotype resembles the SMARD1 phenotype. Like the SMN1 product, IGHMBP2 colocalizes with the RNA-processing machinery in both the cytoplasm and the nucleus. Our results show that IGHMBP2 is the second gene found to be defective in spinal muscular atrophy, and indicate that IGHMBP2 and SMN share common functions important for motor neuron maintenance and integrity in mammals.

Mutations in the Gene Encoding Gap Junction Protein α12 (Connexin 46.6) Cause Pelizaeus-Merzbacher–Like Disease

The hypomyelinating leukodystrophies X-linked Pelizaeus-Merzbacher disease (PMD) and Pelizaeus-Merzbacher-like disease (PMLD) are characterized by nystagmus, progressive spasticity, and ataxia. In a consanguineous family with PMLD, we performed a genomewide linkage scan using the GeneChip Mapping EA 10K Array (Affymetrix) and detected a single gene locus on chromosome 1q41-q42. This region harbors the GJA12 gene, which encodes gap junction protein alpha 12 (or connexin 46.6). Gap junction proteins assemble into intercellular channels through which signaling ions and small molecules are exchanged. GJA12 is highly expressed in oligodendrocytes, and, therefore, it serves as an excellent candidate for hypomyelination in PMLD. In three of six families with PMLD, we detected five different GJA12 mutations, including missense, nonsense, and frameshift mutations. We thereby confirm previous assumptions that PMLD is genetically heterogeneous. Although the murine Gja12 ortholog is not expressed in sciatic nerve, we did detect GJA12 transcripts in human sciatic and sural nerve tissue by reverse-transcriptase polymerase chain reaction. These results are in accordance with the electrophysiological finding of reduced motor and sensory nerve conduction velocities in patients with PMLD, which argues for a demyelinating neuropathy. In this study, we demonstrate that GJA12 plays a key role in central myelination and is involved in peripheral myelination in humans.

Infantile spinal muscular atrophy with respiratory distress type 1 (SMARD1)

Autosomal recessive spinal muscular atrophy with respiratory distress type 1 (SMARD1) is the second anterior horn cell disease in infants in which the genetic defect has been defined. SMARD1 results from mutations in the gene encoding the immunoglobulin μ‐binding protein 2 (IGHMBP2) on chromosome 11q13. Our aim was to review the clinical features of 29 infants affected with SMARD1 and report on 26 novel IGHMBP2 mutations. Intrauterine growth retardation, weak cry, and foot deformities were the earliest symptoms of SMARD1. Most patients presented at the age of 1 to 6 months with respiratory distress due to diaphragmatic paralysis and progressive muscle weakness with predominantly distal lower limb muscle involvement. Sensory and autonomic nerves are also affected. Because of the poor prognosis, there is a demand for prenatal diagnosis, and clear diagnostic criteria for infantile SMARD1 are needed. The diagnosis of SMARD1 should be considered in infants with non‐5q spinal muscular atrophy, neuropathy, and muscle weakness and/or respiratory distress of unclear cause. Furthermore, consanguineous parents of a child with sudden infant death syndrome should be examined for IGHMBP2 mutations.

Escobar Syndrome Is a Prenatal Myasthenia Caused by Disruption of the Acetylcholine Receptor Fetal γ Subunit

Escobar syndrome is a form of arthrogryposis multiplex congenita and features joint contractures, pterygia, and respiratory distress. Similar findings occur in newborns exposed to nicotinergic acetylcholine receptor (AChR) antibodies from myasthenic mothers. We performed linkage studies in families with Escobar syndrome and identified eight mutations within the gamma -subunit gene (CHRNG) of the AChR. Our functional studies show that gamma -subunit mutations prevent the correct localization of the fetal AChR in human embryonic kidney-cell membranes and that the expression pattern in prenatal mice corresponds to the human clinical phenotype. AChRs have five subunits. Two alpha, one beta, and one delta subunit are always present. By switching gamma to epsilon subunits in late fetal development, fetal AChRs are gradually replaced by adult AChRs. Fetal and adult AChRs are essential for neuromuscular signal transduction. In addition, the fetal AChRs seem to be the guide for the primary encounter of axon and muscle. Because of this important function in organogenesis, human mutations in the gamma subunit were thought to be lethal, as they are in gamma -knockout mice. In contrast, many mutations in other subunits have been found to be viable but cause postnatally persisting or beginning myasthenic syndromes. We conclude that Escobar syndrome is an inherited fetal myasthenic disease that also affects neuromuscular organogenesis. Because gamma expression is restricted to early development, patients have no myasthenic symptoms later in life. This is the major difference from mutations in the other AChR subunits and the striking parallel to the symptoms found in neonates with arthrogryposis when maternal AChR auto-antibodies crossed the placenta and caused the transient inactivation of the AChR pathway.

Congenital myopathy with excess of thin myofilaments

Three unrelated young children are reported to have suffered since birth from muscle hypotonia and two of them from fatal respiratory insufficiency. Muscle tissues were found to contain large masses of thin myofilaments, immunologically identified as containing actin, but without further morphological features. These masses of thin filaments were found in different muscles at different occasions in the three children, suggesting a disease-specific morphological and possibly nosological feature all of them justifying classification as congenital myopathy with excess of actin or actin myopathy. The lesions were dissimilar to hyaline bodies in that the latter consist of granular material which is faintly positive for ATPase activity whereas the masses of thin filaments are devoid of ATPase activity. Two of our three patients also had intranuclear rods with virtually no sarcoplasmic rods suggesting the term of this congenital myopathy as actin myopathy with intranuclear rods.

Antioxidative activity of ubiquinol-10 at physiologic concentrations in human low density lipoprotein

Ubiquinol-10 is a powerful lipid-soluble antioxidant found in cell membranes and lipoproteins in vivo. Its mechanism of action on lipid peroxidation has been determined in model and biological systems. Data concerning antioxidative activity of ubiquinol-10 in lipoproteins, however, are still controversial. The present work examines its role in the prevention of low density lipoprotein (LDL) oxidation, specifically its influence on a copper-mediated oxidative modification of human LDL in vitro. We found that ubiquinol-10 incorporated in LDL in subnormal concentrations (0.05-0.13 mol/mol LDL incorporated in comparison with 0.10-1.20 mol/mol LDL reported as normally in human LDL) slightly but not significantly decreased production of lipid peroxidation products (lipid peroxides, conjugated dienes, thiobarbituric acid-reactive substances) during the first hours of oxidation. The extent of apolipoprotein B modification (LDL fluorescence at 360/430 nm) was also decreased. Increasing the ubiquinol-10 concentration in LDL to 0.55-1.48 mol/mol LDL made it significantly more resistant to copper-mediated oxidation than native LDL. Adding the same amounts of either ubiquinone-10 or alpha-tocopherol to the LDL suspension had almost no effect on its oxidation. Ubiquinol-10 decreased alpha-tocopherol consumption during LDL oxidation and was consumed more rapidly than the latter. These results demonstrate that LDL ubiquinol-10 content is an important factor influencing LDL susceptibility to oxidation by copper and suggest that it represents the first line of defense against oxidative modification in human LDL.

X-chromosome methylation in manifesting and healthy carriers of dystrophinopathies: concordance of activation ratios among first degree female relatives and skewed inactivation as cause of the affected phenotypes

The X-chromosome activity states of 11 manifesting carriers of dystrophinopathies, all with normal karyotypes, were estimated by restriction fragment length polymorphism (RFLP)-methylation analysis with the probes M27β (DXS255), p2-19(DXS605) and pSPT/PGK (PGK1) to test the role of skewed X-inactivation ratios as the cause of their affected phenotypes. In eight cases preferential inactivation of the putative X chromosome carrying the normal dystrophin allele in ≥90% of their peripheral lymphocytes was observed, two cases showed non-appparent deviant ratios (60∶40 and 70∶30) from the theoretically expected values around the mean of 50% and in one case the three markers employed yielded no information. The analysis of the X-inactivation ratio in six mother-daughter pairs, all non-manifesting Duchenne muscular dystrophy (DMD) carriers, and in the close female relatives of the patients showed: (a) neither of the two X chromosomes was preferentially inactivated with respect to their parental origin; (b) a high concordance among the activation ratios of mothers and daughters, a result difficult to explain just in terms of random X-chromosome inactivation.

Low density lipoprotein oxidizability by copper correlates to its initial ubiquinol-10 and polyunsaturated fatty acid content

At an early stage of oxidation induced by Cu2+, the rate of oxidative modification of human low density lipoprotein (LDL) from healthy donors correlated negatively to its ubiquinol‐10 (r = −0.58, P < 0.01) and positively to its polyunsaturated fatty acid (PUFA) (r = 0.53, P < 0.05) content. The PUFA/ubiquinol‐10 ratio was the best predictor of LDL susceptibility to oxidation (r = 0.68, P < 0.01). No significant correlation between LDL oxidizability and its α‐tocopherol content was found at any oxidation stage. It is suggested that ubiquinol‐10 plays a central role in the early protection of LDL PUFAs against Cu2+‐induced oxidation whereas α‐tocopherol posesses both pro‐ and antioxidant activity.

Diaphragmatic Spinal Muscular Atrophy with Respiratory Distress Is Heterogeneous, and One Form Is Linked to Chromosome 11q13-q21

To the Editor: Diaphragmatic spinal muscular atrophy (SMA) has been delineated as a variant of infantile SMA (SMA1 [MIM 253300]) (Mellins et al. 1974; Bertini et al. 1989). The most prominent symptoms are severe respiratory distress resulting from diaphragmatic paralysis with eventration shown on chest x-ray and predominant involvement of the upper limbs and distal muscles. In contrast to classic SMA1, in diaphragmatic SMA the upper spinal cord is more severely affected than the lower section. The pmn mouse presents with progressive motor neuronopathy and a disease that closely resembles diaphragmatic SMA (Schmalbruch et al. 1991). The pmn locus has been mapped to murine chromosome 13 (Brunialti et al. 1995). Here we report on nine patients from three families with diaphragmatic SMA following autosomal recessive inheritance. The diagnosis of diaphragmatic SMA was made on the basis of clinical criteria (Rudnik-Schoneborn et al. 1996). Family 1 is of Lebanese origin; family 2, German origin; and family 3, Italian origin. We obtained DNA samples from these families after receiving informed consent, in accordance with the Declaration of Helsinki. In family 1 (fig. 1A), the parents are first cousins. The first affected son died, at the age of 10 wk, of suspected sudden infant death syndrome (SIDS). One daughter presented, at the age of 6 wk, with feeding difficulties and progressive respiratory distress. Chest x-ray showed eventration of the diaphragm. Mechanical ventilation was initiated at the age of 8 wk. She developed progressive muscular atrophy with complete paralysis of the upper and lower limbs and mild contractures of the knee and ankle joints. Three other children, nonidentical twin daughters and the youngest daughter, died of respiratory failure—the twins at the age of 8 and 9 wk and the youngest daughter at the age of 8 wk. Autopsy specimens were taken from gastrocnemius muscle in both twins and from the upper spinal cord in one twin. Skeletal-muscle histology revealed neurogenic atrophy without signs of reinnervation. Ultrastructurally, the motor end plates lacked nerve terminals and showed postsynaptic degenerative changes characterized by deep invaginations. The diameter of anterior spinal roots was reduced in the upper spinal cord. The remaining motor neurons showed chromatolysis. These findings offer two different pathophysiological concepts: (1) degeneration of the anterior horn cells of the spinal cord with neurogenic muscular atrophy suggests dying-forward atrophy, and (2) presynaptic and postsynaptic signs of motor end-plate degeneration suggest dying-back atrophy. In family 2 (fig. 1B), the first child had severe muscular hypotonia and died, at the age of 9 wk, of cardiorespiratory failure. The third child has been mechanically ventilated since the age of 3 mo. In family 3 (fig. 1C), which has been reported in detail elsewhere (Novelli et al. 1995), the gene locus for SMA1, on chromosome 5q, has been excluded. Both affected sibs presented with respiratory insufficiency right after birth and with the typical signs of diaphragmatic SMA. Figure 1 Haplotypes in families with diaphragmatic SMA subtypes. A, Family 1 (Lebanese origin): age at onset, 6–10 wk. B, Family 2 (German origin): age at onset, 9–12 wk. C, Family 3 (Italian origin): onset at birth. Haplotype analysis indicated ... First, we confirmed that, in families 1 and 2, there is no linkage of the trait to markers of the SMA locus on 5q11.2-q13.3, as there is in family 3. Second, the orthologous regions corresponding to the murine pmn gene region on human chromosomes 1q and 7p were excluded as gene loci responsible for the disease (Grohmann et al. 1998). To locate the gene locus for diaphragmatic SMA, a whole-genome scan was undertaken in family 1. Microsatellite analysis was performed, by standard semiautomated methods, by an ABI 377-Sequencer, and the results were processed by GENESCAN software, as described elsewhere (Saar et al. 1997). The whole-genome linkage scan was performed with the use of 340 polymorphic fluorescence–labeled markers spaced at ∼10-cM intervals throughout the autosomal part of the genome. Subsequent fine mapping was performed with eight additional microsatellite markers. Markers were chosen from the Genethon final linkage map. Two-point parametric linkage analyses were performed with the LINKAGE package, version 5.2 (Lathrop and Lalouel 1984), under the following assumptions: a regular, fully penetrant autosomal recessive trait locus with a disease-allele frequency of .002 and no phenocopy rate, codominant marker loci with uniformly distributed allele frequencies, and standard recombination rates. Multipoint analysis was performed with the GENEHUNTER program, version 1.3 (Kruglyak et al. 1996). Genomewide linkage scanning of family 1 revealed linkage of diaphragmatic SMA only to markers on chromosome 11q13-q21. In the following, we name this subtype of diaphragmatic SMA “spinal muscular atrophy with respiratory distress” (SMARD). For the markers D11S1296, D11S4095, D11S901, D11S1358, and D11S1757, a maximum two-point LOD score of 3.16 at recombination fraction (θ) 0 was obtained. The two-point LOD scores for 13 markers on chromosome 11q are summarized in table 1. Haplotype analysis revealed a recombination event in individual 2.4 that placed the disease locus distal to marker D11S1883 (fig. 1A). The crossing-over in individual 2.1 placed the disease locus proximal to marker D11S917. Consistent with parental consanguinity, all affected siblings from family 1 were autozygous for all markers within the cosegregating segment. Multipoint linkage analysis with the use of 13 markers yielded a maximum LOD score of 3.86, which clearly places the disease locus between D11S1883 and D11S917 (Genethon map positions 68.5 cM and 100.9 cM). Table 1 LOD-Score Values at Standard Recombination Rates for Markers on Chromosome 11q in Lebanese Family 1 In family 2, the two affected sibs shared two identical parental haplotypes in the SMARD cosegregating segment on 11q13-q21, a finding that supports the assignment of the SMARD locus to this region (fig. 1B). In family 3, haplotype analysis was inconsistent with linkage to the markers tested (fig. 1C). Thus, this locus was excluded as being responsible for the disease in this family. Our finding that diaphragmatic SMA with onset at age 6–12 wk is linked to chromosome 11q markers in two apparently unrelated families from different countries (families 1 and 2) but that diaphragmatic SMA with onset at birth does not show such linkage (family 3) suggests that diaphragmatic SMA is both clinically and genetically heterogeneous. The prevalence of diaphragmatic SMA is unknown. However, in a series of >200 patients with early-onset SMA, ∼1% presented with diaphragmatic SMA and did not have a deletion of the survival motor-neuron gene (SMN) on chromosome 5q (Rudnik-Schoneborn et al. 1996). Considering the case history of the affected son from family 1 who had suspected SIDS, we presume that some of those infants with SIDS may possibly have been misdiagnosed. We are currently looking for further patients with SMARD, to refine the large cosegregating region on chromosome 11q.

Mutations in MEGF10, a regulator of satellite cell myogenesis, cause early onset myopathy, areflexia, respiratory distress and dysphagia (EMARDD)

Infantile myopathies with diaphragmatic paralysis are genetically heterogeneous, and clinical symptoms do not assist in differentiating between them. We used phased haplotype analysis with subsequent targeted exome sequencing to identify MEGF10 mutations in a previously unidentified type of infantile myopathy with diaphragmatic weakness, areflexia, respiratory distress and dysphagia. MEGF10 is highly expressed in activated satellite cells and regulates their proliferation as well as their differentiation and fusion into multinucleated myofibers, which are greatly reduced in muscle from individuals with early onset myopathy, areflexia, respiratory distress and dysphagia.