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Mutation Analysis of CHRNA1, CHRNB1, CHRND, and RAPSN Genes in Multiple Pterygium Syndrome/Fetal Akinesia Patients

2008; Elsevier BV; Volume: 82; Issue: 1 Linguagem: Inglês

10.1016/j.ajhg.2007.09.016

1537-6605

Julie Vogt, Benjamin J. Harrison, Hayley Spearman, Judy Cossins, Sascha Vermeer, Lambert Naudin ten Cate, Neil V. Morgan, David Beeson, Eamonn R. Maher,

Neonatal Respiratory Health Research

Multiple pterygium syndromes (MPS) comprise a group of multiple congenital anomaly disorders characterized by webbing (pterygia) of the neck, elbows, and/or knees and joint contractures (arthrogryposis). MPS are phenotypically and genetically heterogeneous but are traditionally divided into prenatally lethal and nonlethal (Escobar) types. Previously, we and others reported that recessive mutations in the embryonal acetylcholine receptor g subunit (CHRNG) can cause both lethal and nonlethal MPS, thus demonstrating that pterygia resulted from fetal akinesia. We hypothesized that mutations in acetylcholine receptor-related genes might also result in a MPS/fetal akinesia phenotype and so we analyzed 15 cases of lethal MPS/fetal akinesia without CHRNG mutations for mutations in the CHRNA1, CHRNB1, CHRND, and rapsyn (RAPSN) genes. No CHRNA1, CHRNB1, or CHRND mutations were detected, but a homozygous RAPSN frameshift mutation, c.1177-1178delAA, was identified in a family with three children affected with lethal fetal akinesia sequence. Previously, RAPSN mutations have been reported in congenital myasthenia. Functional studies were consistent with the hypothesis that whereas incomplete loss of rapsyn function may cause congenital myasthenia, more severe loss of function can result in a lethal fetal akinesia phenotype. Multiple pterygium syndromes (MPS) comprise a group of multiple congenital anomaly disorders characterized by webbing (pterygia) of the neck, elbows, and/or knees and joint contractures (arthrogryposis). MPS are phenotypically and genetically heterogeneous but are traditionally divided into prenatally lethal and nonlethal (Escobar) types. Previously, we and others reported that recessive mutations in the embryonal acetylcholine receptor g subunit (CHRNG) can cause both lethal and nonlethal MPS, thus demonstrating that pterygia resulted from fetal akinesia. We hypothesized that mutations in acetylcholine receptor-related genes might also result in a MPS/fetal akinesia phenotype and so we analyzed 15 cases of lethal MPS/fetal akinesia without CHRNG mutations for mutations in the CHRNA1, CHRNB1, CHRND, and rapsyn (RAPSN) genes. No CHRNA1, CHRNB1, or CHRND mutations were detected, but a homozygous RAPSN frameshift mutation, c.1177-1178delAA, was identified in a family with three children affected with lethal fetal akinesia sequence. Previously, RAPSN mutations have been reported in congenital myasthenia. Functional studies were consistent with the hypothesis that whereas incomplete loss of rapsyn function may cause congenital myasthenia, more severe loss of function can result in a lethal fetal akinesia phenotype. Multiple pterygia are found infrequently in children with arthrogryposis and in fetuses with fetal akinesia syndrome.1Hall J.G. Reed S.D. Rosenbaum K.N. Gershanik J. Chen H. Wilson K.M. Limb pterygium syndromes: a review and report of eleven patients.Am. J. Med. Genet. 1982; 12: 377-379Crossref PubMed Scopus (99) Google Scholar Autosomal-recessive inheritance is most common, but autosomal-dominant and X-linked inheritance has been described. Lethal multiple pterygium syndrome (LMPS [MIM 253290]) is an autosomal-recessively inherited disorder characterized by intrauterine growth retardation, multiple pterygia and flexion contractures causing severe arthrogryposis, and fetal akinesia. In severe cases, subcutaneous edema may be severe, causing fetal hydrops with cystic hygroma and lung hypoplasia. Developmental defects including cleft palate, cryptorchidism, intestinal malrotation, cardiac hypoplasia, diaphragmatic hernia, obstructive uropathy, microcephaly, or cerebellar and pontine hypoplasia can also be present.2Hall J.G. The lethal multiple pterygium syndromes.Am. J. Med. Genet. 1984; 4: 803-807Crossref Scopus (63) Google Scholar, 3Froster U.G. Stallmach T. Wisser J. Hebisch G. Robbiani M.B. Huch R. Huch A. Lethal multiple pterygium syndrome: suggestion for a consistent pathological workup and review of reported cases.Am. J. Med. Genet. 1997; 68: 82-85Crossref PubMed Scopus (18) Google Scholar The etiology of MPS is heterogeneous. In a few cases, a diagnosis of a specific primary myopathy, metabolic, or neurodevelopmental disorder is made, but until recently, in many cases the underlying etiology was unknown.4Cox P.M. Brueton L.A. Bjelogrlic P. Pomroy P. Sewry C.A. Diversity of neuromuscular pathology in lethal multiple pterygium syndrome.Pediatr. Dev. Pathol. 2003; 6: 59-68Crossref PubMed Scopus (13) Google Scholar Recently, defects in the embryonal acetylcholine receptor (AChR) were discovered to account for a significant proportion of patients with lethal MPS and fetal akinesia. Thus, mutations in the acetylcholine receptor γ subunit gene (CHRNG) were detected in approximately 30% of lethal MPS cases, and also in nonlethal Escobar variant MPS.5Morgan N.V. Brueton L.A. Cox P. Greally M.T. Tolmie J. Pasha S. Aligianis I.A. van Bokhoven H. Marton T. Al-Gazali L. et al.Mutations in the embryonal subunit of the acetylcholine receptor (CHRNG) cause lethal and Escobar variants of multiple pterygium syndrome.Am. J. Hum. Genet. 2006; 79: 390-395Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 6Hoffmann K. Muller J.S. Stricker S. Megarbane A. Rajab A. Lindner T.H. Cohen M. Chouery E. Adaimy L. Ghanem I. et al.Escobar syndrome is a prenatal myasthenia caused by disruption of the acetylcholine receptor fetal gamma subunit.Am. J. Hum. Genet. 2006; 79: 303-312Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar The nicotinic AChR of skeletal muscle is a pentameric transmembrane protein composed of four different subunits and exists in two forms. The adult form is predominant in innervated adult muscle and the embryonic form predominates in fetal and denervated muscle. The embryonic AChR consists of two alpha and one each beta, gamma, and delta subunit (α2βδγ), whereas in the adult AChR, the gamma subunit is replaced by an epsilon subunit (α2βδɛ).7Mishina M. Takai T. Imoto K. Noda M. Takahashi T. Numa S. Molecular distinction between fetal and adult forms of muscle acetylcholine receptor.Nature. 1986; 321: 406-411Crossref PubMed Scopus (726) Google Scholar, 8Bouzat C. Bren N. Sine S.M. Structural basis of the different gating kinetics of fetal and adult acetylcholine receptors.Neuron. 1994; 6: 1395-1402Abstract Full Text PDF Scopus (129) Google Scholar In humans, the switch from γ to ɛ occurs during fetal life and is apparently complete by 31 weeks gestation (in rodents the switch occurs postnatally).9Hesselmans L.F. Jennekens F.G. Van den Oord C.J. Veldman H. Vincent A. Development of innervation of skeletal muscle fibers in man: relation to acetylcholine receptors.Anat. Rec. 1993; 236: 553-562Crossref PubMed Scopus (99) Google Scholar, 10Kues W.A. Sakmann B. Witzemann V. Differential expression patterns of five acetylcholine receptor subunit genes in rat muscle during development.Eur. J. Neurosci. 1995; 7: 1376-1385Crossref PubMed Scopus (59) Google Scholar, 11Takahashi M. Kubo T. Mizoguchi A. Carlson C.G. Endo K. Ohnishi K. Spontaneous muscle action potentials fail to develop without fetal-type acetylcholine receptors.EMBO Rep. 2002; 3: 674-681Crossref PubMed Scopus (40) Google Scholar The identification of CHRNG mutations in MPS patients suggested that pterygia resulted from early-onset fetal akinesia. Although the embryonal AChR has a key role in normal prenatal muscle development, the γ subunit is not required postnatally and so Escobar variant MPS patients did not demonstrate marked muscle weakness.6Hoffmann K. Muller J.S. Stricker S. Megarbane A. Rajab A. Lindner T.H. Cohen M. Chouery E. Adaimy L. Ghanem I. et al.Escobar syndrome is a prenatal myasthenia caused by disruption of the acetylcholine receptor fetal gamma subunit.Am. J. Hum. Genet. 2006; 79: 303-312Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar Mutations in other AChR subunits (except CHRNE) would be predicted to produce both pre- and postnatal neuromuscular transmission deficits, and we speculated that mutations in genes implicated in embryonal AChR function might cause fetal akinesia and pterygia in patients without CHRNG mutations.6Hoffmann K. Muller J.S. Stricker S. Megarbane A. Rajab A. Lindner T.H. Cohen M. Chouery E. Adaimy L. Ghanem I. et al.Escobar syndrome is a prenatal myasthenia caused by disruption of the acetylcholine receptor fetal gamma subunit.Am. J. Hum. Genet. 2006; 79: 303-312Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar In addition, because we have found that intrafamilial phenotypic variation in kindreds with CHRNG mutations such that some affected children had fetal akinesia without detectable pterygia, we also included patients referred for CHRNG mutation analysis without pterygia. Mutation analysis of CHRNG was undertaken in patients with MPS and/or fetal akinesia as described previously (unpublished data).6Hoffmann K. Muller J.S. Stricker S. Megarbane A. Rajab A. Lindner T.H. Cohen M. Chouery E. Adaimy L. Ghanem I. et al.Escobar syndrome is a prenatal myasthenia caused by disruption of the acetylcholine receptor fetal gamma subunit.Am. J. Hum. Genet. 2006; 79: 303-312Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar Fifteen probands with no evidence of a germline CHRNG mutation were then selected for mutation analysis of the coding sequence and flanking intronic sequence of CHRNA1, CHRNB1, CHRND, and RAPSN. The clinical features of the 15 families are summarized in Table 1.Table 1Clinical Features of 15 Fetuses AnalyzedFamily IDEthnic OriginParental ConsanguinityPhenotypeMPS013PakistaniyesLMPSMPS012BengaliyesAMC, no pterygiaMPS014whitenoLMPSMPS016whitenoLMPSMPS017whitenoLMPSMPS018whitenoLMPSMPS019IndianyesLMPSMPS020whitenoLMPSMPS021whitenoLMPSMPS022AfrikanernoLMPSMPS023whitenoLMPSMPS010PakistaniyesAMC, no pterygiaMPS009PakistaniyesLMPSMPS024PalestinianyesLMPSMPS025EthiopiannoLMPSLMPS, lethal multiple pterygium syndrome. Open table in a new tab LMPS, lethal multiple pterygium syndrome. DNA was extracted and mutation analysis was performed by standard methods and conditions (details of primer sequences available on request). No pathogenic mutations were detected in CHRNA1, CHRNB1, or CHRND. However, a RAPSN frameshift mutation was detected in a consanguineous family with three affected children (see Figure 1). Both parents were heterozygous, and all three affected children were homozygous, for a c.1177-1178delAA mutation (see Figure 1). The family presented when fetal akinesia sequence was detected at 19 weeks gestation in twin male fetuses. On ultrasound examination, both fetuses had micrognathia and fixed position of the hands, elbows, and feet. There were no respiratory movements, the thorax appeared small, and there was mild hydrops. The pregnancy was terminated at 23 weeks of gestation. Postmortem examination revealed monochorionic, monoamniotic twins with no evidence of growth retardation (twin 1, 0.587 kg [∼50 percentile]; twin 2, 0.665 kg [>50 percentile]). Both twins had a flat facial profile, hypertelorism, moderate micrognathia, and a short broad neck. They also had mild hydrops with hydrothoraces and subcutaneous edema of the head, neck, back of the shoulders, and proximal extremities (Figures 2A and 2B). The lungs were hypoplastic and the diaphragm was thin and membranous. Both twins displayed an abnormal posture, with hyperextension at the wrists and flexion of the fingers. Ankles were fixed in a varus position and the toes were hyperextended. No pterygia were detected. Macroscopic and microscopic examination of the brain showed no abnormality. Muscle histopathology revealed muscle fibers of varying diameters (considered to be normal for the gestational age) with a normal striated pattern and no fibrosis. Chromosome analysis was normal and molecular testing for congenital myotonic dystrophy was negative. The couple went on to have another similarly affected female singleton pregnancy. Fetal akinesia sequence was detected on ultrasound at 19 weeks of gestation and the pregnancy was terminated at 23 weeks gestation. Postmortem examination showed fetal dysmorphology (micrognathia, low-set ears, a short nose, a short philtrum, thin lips, and a short broad neck) and subcutaneous edema of the head, neck, and shoulders (Figure 2C). The elbows were abnormally flexed, the wrists were hyperextended, and the second and third digits overlapped on both hands. No pterygia were identified. The lungs were hypoplastic. Neuromuscular histopathological examination failed to identify a specific underlying cause for the fetal akinesia. The brain and spinal cord were normal on macroscopic and microscopic examination. The muscle fibers were of normal diameter for gestational age. No abnormalities were detected on enzyme analysis. On electron microscopy, the striated muscle appeared well developed. All three cases showed occasional muscle fibers with centrally located nuclei, but this appearance was observed in <5% of the muscle fibers examined and was not diagnostic of a myotubular dystrophy. The couple also have two healthy children.Figure 2Clinical Features of Fetal Akinesia Associated with Homozygous Rapsyn MutationShow full caption(A and B) Affected twin fetuses.(C) Affected singleton fetus with clinical features of the fetal akinesia sequence.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A and B) Affected twin fetuses. (C) Affected singleton fetus with clinical features of the fetal akinesia sequence. Previously, mutations in AChR subunits have been associated with congenital myasthenic syndromes (CMS). These are a heterogeneous group of inherited disorders of neuromuscular transmission characterized by fatiguable muscle weakness. CMS affecting the AChR may alter AChR channel kinetics or severely reduce the number of AChRs in the postsynaptic membrane and can result in AChR deficiency from mutations in CHRNA1, CHRNB1, CHRND, or CHRNE.12Engel A.G. Ohno K. Shen X.M. Sine S.M. Congenital myasthenic syndromes: multiple molecular targets at the neuromuscular junction.Ann. N Y Acad. Sci. 2003; 998: 138-160Crossref PubMed Scopus (45) Google Scholar, 13Jurkat-Rott K. Lehmann-Horn F. Muscle channelopathies and critical points in functional and genetic studies.J. Clin. Invest. 2005; 115: 2000-2009Crossref PubMed Scopus (76) Google Scholar However, mutations in CHRNE generally result in a mild phenotype, whereas AChR deficiency mutations in CHRNA1, CHRNB1, and CHRND are rare and more severe. Although the CMS phenotypes are distinct from that of MPS, one CMS patient who was a compound heterozygote for a mutation that gives rise to fast-channel AChR kinetics and a null mutation in CHRND had arthrogryposis.14Brownlow S. Webster R. Croxen R. Brydson M. Neville B. Lin J.P. Vincent A. Newsom-Davis J. Beeson D. Acetylcholine receptor delta subunit mutations underlie a fast-channel myasthenic syndrome and arthrogryposis multiplex congenita.J. Clin. Invest. 2001; 108: 125-130Crossref PubMed Scopus (72) Google Scholar CMS may also result from mutations in other postsynaptic proteins involved in neuromuscular transmission or in the formation and maintenance of the neuromuscular junction. Thus, recessive germline mutations in RAPSN have been reported in patients with CMS and AChR deficiency.15Ohno K. Engel A.G. Shen X.M. Selcen D. Brengman J. Harper C.M. Tsujino A. Milone M. Rapsyn mutations in humans cause endplate acetylcholine-receptor deficiency and myasthenic syndrome.Am. J. Hum. Genet. 2002; 70: 875-885Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 16Burke G. Cossins J. Maxwell S. Owens G. Vincent A. Robb S. Nicolle M. Hilton-Jones D. Newsom-Davis J. Palace J. Beeson D. Rapsyn mutations in hereditary myasthenia: distinct early- and late-onset phenotypes.Neurology. 2003; 61: 826-828Crossref PubMed Scopus (109) Google Scholar, 17Maselli R.A. Dunne V. Pascual-Pascual S.I. Bowe C. Agius M. Frank R. Wollmann R.L. Rapsyn mutations in myasthenic syndrome due to impaired receptor clustering.Muscle Nerve. 2003; 28: 293-301Crossref PubMed Scopus (33) Google Scholar, 18Muller J.S. Mildner G. Muller-Felber W. Schara U. Krampfl K. Petersen B. Petrova S. Stucka R. Mortier W. Bufler J. et al.Rapsyn N88K is a frequent cause of congenital myasthenic syndromes in European patients.Neurology. 2003; 60: 1805-1810Crossref PubMed Scopus (74) Google Scholar The RAPSN gene product (rapsyn, receptor-associated protein of the synapse) has a key role in the clustering of AChRs at the neuromuscular synapse.19Apel E.D. Roberds S.L. Campbell K.P. Merlie J.P. Rapsyn may function as a link between the acetylcholine receptor and the agrin-binding dystrophin-associated glycoprotein complex.Neuron. 1995; 15: 115-126Abstract Full Text PDF PubMed Scopus (197) Google Scholar The most frequent RAPSN mutation associated with CMS is the founder p.N88K missense mutation that is thought to have arisen in an ancient Indo-European population.20Muller J.S. Abicht A. Burke G. Cossins J. Richard P. Baumeister S.K. Stucka R. Eymard B. Hantai D. Beeson D. Lochmuller H. The congenital myasthenic syndrome mutation RAPSN N88K derives from an ancient Indo-European founder.J. Med. Genet. 2004; 41: e104Crossref PubMed Scopus (30) Google Scholar Genotype-phenotype correlations have been described for CMS-associated RAPSN mutations, such that individuals who are homozygous for a p.N88K disease have less severe disease than compound heterozygotes with a single p.N88K allele.21Dunne V. Maselli R.A. Identification of pathogenic mutations in the human rapsyn gene.J. Hum. Genet. 2003; 48: 204-207Crossref PubMed Scopus (32) Google Scholar These observations suggested that p.N88K may cause a less severe defect than mutations that produce a truncated protein or altered membrane attachment. To date, homozygosity (or compound heterozygosity) for mutations in RAPSN predicted to cause a truncated protein have not been described in CMS. The c.1177-1178delAA mutation we identified was not detected in 340 control chromosomes (140 in the current study and 200 reported previously16Burke G. Cossins J. Maxwell S. Owens G. Vincent A. Robb S. Nicolle M. Hilton-Jones D. Newsom-Davis J. Palace J. Beeson D. Rapsyn mutations in hereditary myasthenia: distinct early- and late-onset phenotypes.Neurology. 2003; 61: 826-828Crossref PubMed Scopus (109) Google Scholar) and has previously been reported (in combination with the p.N88K allele) in patients with CMS. Affected patients had decreased fetal movements, flexion contractures, craniofacial anomalies, hypotonia, and intermittent respiratory failure.16Burke G. Cossins J. Maxwell S. Owens G. Vincent A. Robb S. Nicolle M. Hilton-Jones D. Newsom-Davis J. Palace J. Beeson D. Rapsyn mutations in hereditary myasthenia: distinct early- and late-onset phenotypes.Neurology. 2003; 61: 826-828Crossref PubMed Scopus (109) Google Scholar, 22Banwell B.L. Ohno K. Sieb J.P. Engel A.G. Novel truncating RAPSN mutations causing congenital myasthenic syndrome responsive to 3,4-diaminopyridine.Neuromuscul. Disord. 2004; 14: 202-207Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar We postulated that the c.1177-1178delAA mutation caused a more severe deficit in rapsyn function than p.N88K and so homozygosity for c.1177-1178delAA caused a fetal akinesia, rather than a CMS phenotype. To test this hypothesis, the effect of the c.1177-1178delAA mutation on rapsyn function was investigated. The c.1177-1178delAA mutation predicts a frameshift after residue 392 of rapsyn resulting in 82 C-terminal missense amino acids (Figure 3A). Wild-type and mutant rapsyn were tagged with EGFP in-frame at their C termini and cotransfected with cDNAs encoding the human AChR into the muscle cell line TE671. TE671 cells were maintained at 37°C in DMEM (Sigma-Aldrich) supplemented with 10% FCS (TCS Cellworks) and 100 units/ml each of penicillin G and streptomycin (PS) purchased from Invitrogen. TE671 cells do not express detectable levels of endogenous rapsyn polypeptide. After transfection, effects on AChR clustering were studied. Thus, TE671 cells were seeded at 2 × 105 cells per well on coverslips in 6-well plates and the following day were transfected with a total of 3 μg DNA per well via calcium phosphate precipitation. Amounts of DNA used per well was 1 μg AChR α-subunit DNA, 0.5 μg each of AChR β-, δ-, and ɛ-subunit cDNA, and 0.5 μg of rapsyn cDNA or pcDNA3.1-hygro for "no rapsyn" control transfections. Transfections with only rapsyn and no AChR were carried out with 3 μg/well of rapsyn expression plasmids. The next day, the media was replaced with fresh medium. Whereas transfection with wild-type rapsyn resulted in the colocalization of rapsyn and AChR on the cell surface in small dense clusters, transfection with rapsyn-392+82-EGFP resulted in low levels of EGFP fluorescence within the cells and unclustered AChR expressed uniformly along the cell surface. To examine this observation further, we performed a time course of rapsyn expression after transfection by western blots on cell extracts. Confirming the observation from fluorescence microscopy, the mutant rapsyn was present at much lower levels and by 3 days after transfection could not be detected. The results suggest that the mutant rapsyn is rapidly degraded. It is likely that in the patient fetal AChR are not clustered because of the loss of rapsyn, which results in the disruption of the neuromuscular synapse formation and neuromuscular transmission, which in turn results in severe fetal akinesia. We found that homozygosity for a frameshift RAPSN mutation that severely impaired protein stability was associated with a fetal akinesia phenotype. Previously it was reported that rapsyn missense mutations p.L361R and p.N88K showed a partial loss of function with reduced colocalization. Both mutations could effectively mediate agrin-induced AChR clusters in myotubes but they were less stable than clusters generated with wild-type rapsyn.23Cossins J. Burke G. Maxwell S. Spearman H. Man S. Kuks J. Vincent A. Palace J. Fuhrer C. Beeson D. Diverse molecular mechanisms involved in AChR deficiency due to rapsyn mutations.Brain. 2006; 129: 2773-2783Crossref PubMed Scopus (40) Google Scholar This observation is consistent with the hypothesis that RAPSN mutations may cause a spectrum of phenotypes ranging from later onset (third decade) CMS with "mild mutations" through neonatal CMS with arthrogryposis, to fetal akinesia with lethality (homozygosity for severe truncating mutations).24Burke G. Cossins J. Maxwell S. Robb S. Nicolle M. Vincent A. Newsom-Davis J. Palace J. Beeson D. Distinct phenotypes of congenital acetylcholine receptor deficiency.Neuromuscul. Disord. 2004; 14: 356-364Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar The c.1177-1178delAA mutation identified in our family had previously been reported in CMS, although it is not known whether this represents a recurrent or founder mutation. Although the development of neuromuscular junction differs in mice and men, the phenotype of Chrng and Rapsn gene inactivation in mice are similar. Thus, homozygous mutant transgenic mice with targeted disruption of the Rapsn gene died within hours of birth with respiratory insufficiency and profound muscle weakness.25Gautam M. Noakes P.G. Mudd J. Nichol M. Chu G.C. Sanes J.R. Merlie J.P. Failure of postsynaptic specialization to develop at neuromuscular junctions of rapsyn-deficient mice.Nature. 1995; 377: 232-236Crossref PubMed Scopus (464) Google Scholar The mutant mice neuromuscular junction showed no detectable AChR clusters along the length of muscle fibers. Together with the previous reports of CHRNG in MPS, it suggests that functional AChR deficiency will be a significant cause of MPS/fetal akinesia.5Morgan N.V. Brueton L.A. Cox P. Greally M.T. Tolmie J. Pasha S. Aligianis I.A. van Bokhoven H. Marton T. Al-Gazali L. et al.Mutations in the embryonal subunit of the acetylcholine receptor (CHRNG) cause lethal and Escobar variants of multiple pterygium syndrome.Am. J. Hum. Genet. 2006; 79: 390-395Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 6Hoffmann K. Muller J.S. Stricker S. Megarbane A. Rajab A. Lindner T.H. Cohen M. Chouery E. Adaimy L. Ghanem I. et al.Escobar syndrome is a prenatal myasthenia caused by disruption of the acetylcholine receptor fetal gamma subunit.Am. J. Hum. Genet. 2006; 79: 303-312Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar Although we did not identify germline mutations in CHRNA1, CHRNB1, and CHRND in our cohort of patients, further analysis of these genes and of other genes implicated in CMS, such as MUSK26Chevessier F. Faraut B. Ravel-Chapuis A. Richard P. Gaudon K. Bauché S. Prioleau C. Herbst R. Goillot E. Ioos C. et al.MuSK, a new target for mutations causing congenital myasthenic syndrome.Hum. Mol. Genet. 2004; 13: 3229-3240Crossref PubMed Scopus (151) Google Scholar or DOK7,27Beeson D. Higuchi O. Palace J. Cossins J. Spearman H. Maxwell S. Newsom-Davis J. Burke G. Fawcett P. Motomura M. et al.Dok-7 mutations underlie a neuromuscular junction synaptopathy.Science. 2006; 313: 1975-1978Crossref PubMed Scopus (217) Google Scholar may further expand the role of defective signal transmission at the neuromuscular junction in the MPS/fetal akinesia sequence. We thank WellChild and the Wellcome Trust, MRC and Myasthenia Gravis Association/Muscular Dystrophy Campaign for financial support, and we thank the families and referring clinicians for their help with this research. The URLs for data presented herein are as follows:Ensembl Genome Browser, http://www.ensebl.org/Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim/ (for LMPS and EVMPS) Data have been deposited in the Ensemble Genome Browser with the following accession numbers: for human CHRNA1, ENSG00000138435; for CHRNB1, ENSG00000170175; for CHRND, ENSG00000135902; for CHRNG, ENSG00000196811; and for RAPSN, ENSG00000165917.