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, 79 (2), 303-12

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


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

Katrin Hoffmann et al. Am J Hum Genet.


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.


Figure  1.
Figure 1.
Structure and subunit composition of the fetal and adult AChR at muscle cells. Acetylcholine release from nerve terminals results in activation of the AChR at the postsynaptic membrane. This triggers an end-plate potential that activates voltage-dependent sodium channels and finally generates an action potential in the muscle. An AChR consists of a pentamer of paralogous subunits. Two types of skeletal-muscle AChR are identified by their different functions and subunit compositions. A, Fetal AChR. A fetal type of AChR has 2α, β, γ, and δ subunits and is synthesized before week 33 of gestation in humans and before postnatal day P9 in mice. B, Adult AChR. Adult-type AChRs are formed through a gradual replacement of the fetal γ by the adult ɛ subunit.,
Figure  2.
Figure 2.
Pedigrees of families with Escobar syndrome caused by CHRNG mutations
Figure  3.
Figure 3.
Clinical phenotype of patients with Escobar syndrome. The consistent major signs of Escobar syndrome are multiple contractures (arthrogryposis) and multiple pterygia. We show hand contractures (A), rocker-bottom feet with prominent heels (B), and an elbow web with muscular atrophy (C). The phenotype is variable, as shown for two patients from different families. Patient IV-5, from family EG-6, with homozygous mutation γR217C is more severely affected (D) than patient V-1, from family EG-5, with homozygous mutation γ1249G→C (E). The general appearance—with elongated face, small mouth with downturned corners, mild ptosis, down-slanting palpebral fissures, multiple pterygia, and muscular hypotrophy—is present in both patients. Scoliosis is severe in one patient (D) and absent in the other (E).
Figure  4.
Figure 4.
Schematic structure of the AChR γ subunit and localization of the identified mutations. The γ subunit and the other subunits contain an extracellular large N-terminus where acetylcholine binds and drugs and toxins dock (nicotine, muscle relaxants, d-tubocurarine, and α-bungarotoxin). Then, four transmembrane domains (M1–M4) follow with a large cytoplasmic domain (CD2) between M3 and M4 and an extracellular short C-terminus., Subunit structure was adapted from the work of Engel and Sine. The γ subunit consists of 517 aa, starting with the start methionine. Residues were counted, in accordance with traditional nomenclature, from the first amino acid following the signal peptide. S-S marks an important cysteine loop.
Figure  5.
Figure 5.
Evolutionary conservation of residues relevant for identified missense and duplication mutations. A, Evolutionary conservation of residue γR64 relevant for mutation γR64C. The strongly basic residue arginine R is conserved in human β, δ, γ, and ɛ subunits, as well as among vertebrate γ subunits. Furthermore, insertion of an additional cysteine residue instead of arginine might interfere with the cysteine loops relevant in and between AChR subunits. B, Evolutionary conservation of residues relevant for mutation γ78dup(3). The duplication of residues WVL is located before an R that is evolutionarily completely conserved among γ subunits of all species analyzed and close to a conserved W_PDI_L-motif. The analogous position in the ɛ subunit ɛL78 was shown to locate at the interface of α and ɛ subunits in snail AChR and seems to provide a hydrogen bond between the β sheets involved in acetylcholine binding., C, Evolutionary conservation of residue γR217 relevant for mutation γR217C. Residue γR217 is completely conserved in all human subunits as well as within all species analyzed. Furthermore, insertion of an additional cysteine residue instead of arginine might interfere with the cysteine loops relevant in and between AChR subunits. The line marked “M1” depicts the beginning of the transmembrane domain M1. GenBank accession numbers for alignment of human AChR subunits are as follows: NP_000070.1 (CHRNA1), NP_000738.2 (CHRNB1), NP_000742.1 (CHRND), NP_000071.1 (CHRNE), and NP_005190.4 (CHRNG). GenBank accession numbers for interspecies comparison of γ subunit homologs are as follows: NP_005190.4 (Homo sapiens), P13536 (Bos taurus), P04760 (Mus musculus), P18916 (Rattus norvegicus), P02713 (Gallus gallus), P05376 (Xenopus laevis), P02714 (Torpedo californica), Q4RV81 (Tetraodon nigroviridis), Q7T2Y7 (Fugu rubripes), F09E8.7* (Caenorhabditis elegans), and P04755* (Drosophila melanogaster). The Ensembl number is ENSDART00000028118** (Danio rerio). (Those in the figure marked with an asterisk [*] are the closest homologs to the human CHRNG found in D. melanogaster and C. elegans, respectively.)
Figure  6.
Figure 6.
AChR expression. Altered or missing γ subunit prevents surface expression of fetal AChRs in HEK-cell studies. Transfection with the α, β, γ, and δ subunits results in regular assembly and positioning of the fetal AChR at the cellular surface (A). No AChR surface expression is seen when the γ transfection vector carries mutations γ78dup(3) (C), γR217C (E), or γR448X (G) or is completely missing (I). There might be a partial AChR subunit assembly within the cell, since after-permeabilization bungarotoxin stains the ER (D, F, H, and J). (Receptors were visualized with bungarotoxin staining.)
Figure  7.
Figure 7.
In situ hybridization. In situ hybridization at mouse E14.5 shows a significant expression of the γ subunit in skeletal muscles of the limbs, neck, and head, paravertebrally as well as in the diaphragm (A–C). This corresponds to the major sites of the phenotype observed in human Escobar syndrome. Postnatally, the γ subunit expression decreases (D and E), with reciprocal increase of the ɛ subunit (F and G). EOM = Extraocular muscles; ICM = intercostal muscles; PVT = paravertebral trunk.

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