Mutations in its gene may cause congenital myasthenic syndromes. ... might be frequent in European congenital myasthenic syndrome patients of Gypsy ethnic ...
A common mutation (e1267delG) in congenital myasthenic patients of Gypsy ethnic origin A. Abicht, MD; R. Stucka, PhD; V. Karcagi, PhD; A. Herczegfalvi, MD; R. Horva´th, MD; W. Mortier, MD; U. Schara, MD; V. Ramaekers, MD; W. Jost, MD; J. Brunner, MD; G. Janßen, MD; U. Seidel, MD; B. Schlotter, MD; W. Mu¨ller-Felber, MD; D. Pongratz, MD; R. Ru¨del, PhD; and H. Lochmu¨ller, MD
Article abstract—Objective: Mutation analysis of the acetylcholine receptor (AChR) e subunit gene in patients with sporadic or autosomal recessive congenital myasthenic syndromes (CMS). Background: The nicotinic AChR of skeletal muscle is a neurotransmitter-gated ion channel that mediates synaptic transmission at the vertebrate neuromuscular junction. Mutations in its gene may cause congenital myasthenic syndromes. A recently described mutation in exon 12 of the AChR e subunit (e1267delG) disrupts the cytoplasmic loop and the fourth transmembrane region (M4) of the AChR e subunit. Methods: Forty-three CMS patients from 35 nonrelated families were clinically classified as sporadic cases of CMS (group III according to European Neuromuscular Centre consensus) and were analyzed for e1267delG by PCR amplification and sequence analysis. Results: The authors report the complete genomic sequence and organization of the gene coding for the e subunit of the human AChR (accession number AF105999). Homozygous e1267delG was identified in 13 CMS patients from 11 independent families. All e1267delG families were of Gypsy or southeastern European origin. Genotype analysis indicated that they derive from a common ancestor (founder) causing CMS in the southeastern European Gypsy population. Phenotype analysis revealed a uniform pattern of clinical features including bilateral ptosis and mild to moderate fatigable weakness of ocular, facial, bulbar, and limb muscles. Conclusions: The mutation e1267delG might be frequent in European congenital myasthenic syndrome patients of Gypsy ethnic origin. In general, patients (e1267delG) were characterized by the onset of symptoms in early infancy, the presence of ophthalmoparesis, positive response to anticholinesterase treatment, and the benign natural course of the disease. Key words: Congenital myasthenic syndrome—Acetylcholine receptor e subunit—Neuromuscular junction—Neuropediatrics. NEUROLOGY 1999;53:1564–1569
The nicotinic acetylcholine receptor (AChR) of skeletal muscle is a neurotransmitter-gated ion channel that mediates synaptic transmission at the neuromuscular junction.1 The binding of acetylcholine to its receptor gives rise to the opening of a cationselective channel. The resulting ion current depolarizes the muscle membrane, leading to muscle contraction. The channel protein exists in two functionally different forms.2,3 In fetal and denervated muscle, the AChR is composed of the subunits a2, b, g, and d in a molar stoichiometry, whereas in adult muscle, the g subunit is replaced by the e subunit. Each of the five subunits contains four transmembrane domains, which together form a pentameric structure around a central ion channel. The acetylcholine binding sites are at the interfaces between a
and d and between a and g/e. 1 Each subunit is encoded by a distinct gene whose coding sequences are well characterized.4-8 There is extensive homology between subunits and across species. The cDNA sequence for the human e subunit was published by Beeson et al. in 1993,5 but its genomic sequence has not been reported. The AChR of skeletal muscle is involved in several human neuromuscular disorders. In myasthenia gravis, an autoimmune disorder, antibodies are directed against the AChR. By contrast, autoimmune features are absent in the congenital myasthenic syndromes (CMS). In this heterogeneous group of disorders, neuromuscular transmission is impaired by various inherited defects.9 The CMS are defined by clinical, morphologic, and electrophysiologic crite-
From the Genzentrum und Friedrich-Baur-Institut (Drs. Abicht, Stucka, Horva´th, Schlotter, Mu¨ller-Felber, Pongratz, and Lochmu¨ller), LMU Mu¨nchen, Germany; the Department of Biochemistry (Dr. Karcagi), National Institute of Environmental Health, Budapest; Heim Pal Pediatric Hospital (Dr. Herczegfalvi), Budapest, Hungary; Universita¨tskinderklinik und Neuromuskula¨res Labor (Drs. Mortier and Schara), Ruhr-Universita¨t Bochum, Germany; the Neuropa¨diatrie (Dr. Ramaekers), Rheinisch-Westfa¨lische Technische Hochschule Aachen, Germany; the Universita¨tsklinik fu¨r Kinder und Jugendmedizin (Drs. Jost and Brunner), Homburg/Saar, Germany; the Neuropa¨diatrie (Dr. Janßen), Universita¨tskinderklinik, Heinrich-Heine Universita¨t, Du¨sseldorf, Germany; the Klinik fu¨r Pa¨diatrie mit Schwerpunkt Neurologie (Dr. Seidel), Universita¨tsklinikum Charite´, Berlin, Germany; and the Abt. fu¨r Allgemeine Physiologie (Dr. Ru¨del), Universita¨t Ulm, Germany. Supported by grants of the Deutsche Myasthenie Gesellschaft e.V. and the Deutsche Gesellschaft fu¨r Muskelkranke e.V. (H.L.), and a postdoctoral scholarship by the Fritz Thyssen Stiftung (Az. 212 98006) (A.A). Presented in part at the 5th German Workshop of Neurogenetics; Freiburg, Germany; October 22–24, 1998; and the 51st American Academy of Neurology annual meeting; Toronto, Canada; April 17–24, 1999. Received January 12, 1999. Accepted in final form May 17, 1999. Address correspondence and reprint requests to Dr. Hanns Lochmu¨ller, Genzentrum Mu¨nchen, Feodor-Lynen-Str. 25, 81377 Mu¨nchen, Germany. 1564
Copyright © 1999 by the American Academy of Neurology
ria, revealing presynaptic and postsynaptic abnormalities that affect the release10 or size11 of the acetylcholine quanta, the acetylcholinesterase activity,12,13 or function or number of the AChRs.14-16 Substantial progress in the understanding of the pathologic mechanism of the postsynaptic CMS was made by the identification of mutations in various AChR subunit genes.17-31 In most cases of CMS, postsynaptic functions seem to be affected. Mutations causing kinetic abnormalities of the AChR have been reported in several structural domains at strategically important sites, including regions that contribute to the channel pore (M2 domains of the a, b, d, and e subunit) and the acetylcholine binding site (extracellular domain of the a subunit).20-25 Point mutations in these regions change the electrophysiologic properties of the AChR ion channel, leading to the so-called slow-channel CMS.20-25 In general, transmission of the slowchannel CMS is autosomal dominant. Other genetically defined disorders causing kinetic abnormalities of the AChR include fast-channel syndromes and mode-switching kinetics.17 By contrast, CMS resulting from a deficiency of AChR at the end plate are caused by loss-of-function mutations of the e subunit gene.26-30 These mutations give rise to forms of CMS that are transmitted as autosomal recessive traits. Many of these e subunit deficiencies were found to result from “private” mutations, detected in single kinships only. The clinical phenotype that they produce may vary greatly. Here we report the complete genomic sequence of the AChR e subunit gene. Information on intron sequences enable screening for mutations in the entire coding region of the AChR e subunit gene. In this study, 43 CMS patients from 35 nonrelated families were studied, and e1267delG was found in 13 patients from 11 families, allowing for genotype–phenotype correlation. Patients and methods. Patients and DNA samples. Forty-three CMS patients from 35 nonrelated families were included in this study. Their ages varied from 0.5 to 34 years (21 female, 22 male patients). The patients were diagnosed as having sporadic CMS group III according to the European Neuromuscular Centre consensus.32 All patients had presented with symptoms of a myasthenic syndrome within the first years of life (mandatory inclusion criteria). Definite exclusion criteria were the presence of anti-AChR antibodies and a positive response to plasma exchange–immunosuppressive treatment. Additional data, such as an affected sibling, supported the diagnosis of CMS in eight families. All patients were seen by one of the authors, who are experienced pediatric neurologists in Germany and Austria (13 patients) or Hungary (5 patients). About 50% of the patients seen in Germany and Austria were not of German ethnic origin but originated from different southern European countries, which represent the most important immigration countries for Germany. Data concerning clinical, laboratory, and electrophysiologic findings were collected for each patient. Venous blood samples were obtained from the patients and
from their affected and unaffected family members, when available. Genomic DNA was prepared from 10 mL of peripheral blood using a blood and tissue culture DNA extraction kit according to the manufacturer’s recommendations (Qiagen, Hilden, Germany). To determine the intron sequences of the AChR e subunit gene, PCR amplification was performed on genomic DNA of a healthy control individual. Screening for the mutation e1267delG in exon 12 of the AChR e subunit gene was performed by restriction digest on PCR products of all CMS patients. In patients harboring e1267delG, all 12 exons of the e subunit gene were amplified by PCR and sequenced. Electrophysiologic studies. Electromyographic studies were performed by uniform and specified criteria. Repetitive stimulation of two distal and two proximal muscles at a rate of 3 Hz was carried out. In patients with negative results for a decremental response, a single-fiber electromyography was performed to detect a block of neuromuscular transmission. Polymerase chain reaction. PCR conditions were optimized by varying Mg21 concentration and pH and by adding 10% dimethyl sulfoxide. A typical PCR reaction included reaction buffer, 1.25 mmol/L of each primer, and 10 mmol/L of PCR nucleotide mix (Boehringer Mannheim, Germany), with 1 mg of genomic DNA and 2.5 U of Taq DNA polymerase (Boehringer Mannheim, Germany) in a 50-mL reaction volume. A typical cycling protocol comprised (1) denaturation at 94 °C for 5 minutes; (2) 35 cycles of melting at 94 °C for 1 minute, annealing at 50 °C for 1 minute, and extension at 72 °C for 1 minute; and (3) final extension for 7 minutes. Samples were loaded on a 2% agarose gel. Primers. To determine intron sequences, the published cDNA sequences of the human AChR e subunit were used.5 Primers were designed adjacent to putative intron boundaries as predicted by mouse intron– exon boundaries.33 Primer positions are available on request. Information on intronic sequences was obtained by direct sequencing of the PCR-amplified intron regions in both directions. For mutation analysis of a CMS patient, all 12 exons and adjacent splice donor and acceptor sequences of the AChR e subunit gene were amplified using the PCR primers according to the intron sequences. Restriction digest. To detect the mutation e1267delG, a 550/549 base pair (bp) fragment containing exons 11 and 12 was amplified by PCR using primers 59-cacggagcgagctcgtgtttga-39 and 59-ctggagatgggtgggaaattg-39. The e1267delG mutation results in loss of the XagI site. The mutant allele remains undigested, whereas the wild-type allele yields two fragments (355 bp and 195 bp). Restriction enzyme digestion was carried out at 37° C for 4 hours by adding 15 units of XagI in 20 mL of reaction mixture. Restriction fragments were size-fractionated on a 2% agarose gel containing ethidium bromide. Sequence analysis. PCR-amplified fragments of genomic DNA were purified from agarose gel using the Qiagen gel extraction kit according to manufacturer’s recommendations. Sequence analysis was performed directly on PCR products. DNA fragments (each sense and antisense) were sequenced with an Applied Biosystems model 377 DNA sequencer and fluorescein-labeled dideoxy terminators (Perkin-Elmer; Foster City, CA). Nucleotide seOctober (2 of 2) 1999 NEUROLOGY 53 1565
Table 1 Comparison of intron length of the AChR e subunit in different species (human, mouse, rat) Intron Species
1
2
3
4
Human
254 bp
Mouse
164 bp
Rat
225 bp
5
6
7
8
9
10
11
126 bp
123 bp
129 bp
306 bp
334 bp
82 bp
1,210 bp
83 bp
105 bp
99 bp
448 bp
257 bp
77 bp
1,178 bp
83 bp
90 bp
109 bp
74 bp
84 bp
79 bp
72 bp
101 bp
100 bp
444 bp
272 bp
77 bp
1,175 bp
74 bp
80 bp
78 bp
AChR 5 acetylcholine receptor; bp 5 base pairs.
quence was analyzed and compared with the published cDNA sequence of the human AChR e subunit. Exon– intron junctions were positioned according to the G-T/A-G rule. Intron sequences were analyzed for potential proteinbinding sites and enhancer elements using the MatInspector V2.2 software.34 Nucleotides and codons are numbered starting with the first amino acid of the mature peptide, according to the previously published cDNA sequence of the AChR e subunit.5 Genotype analysis. Six polymorphic microsatellite markers on chromosome 17p13 flanking the AChR epsilon subunit gene (D17S849, D17S926, D17S1828, D17S1810, D17S938, and D17S786) were chosen based on information obtained from Genome Database and Ge´ne´thon online services. Markers were amplified by PCR using fluoresceinlabeled primers; size of PCR products was determined on sequencing gels using the GeneScan and GenoTyper software according to the manufacturer’s recommendations (Perkin Elmer Biosystems, Norwalk, CT). Results. Gene sequence of the AChR e subunit. In this study, we report the complete sequence of the human muscle AChR epsilon subunit gene (GenBank accession number AF105999). Nucleotide sequence analysis and comparison with the published cDNA sequence5 revealed the exon–intron structure of the human AChR e subunit gene. The protein-coding region consists of 12 exons divided by 11 introns (table 1). The number of exons and the locations of the intron– exon boundaries correspond well to those reported for the homologous rat and mouse genes33,35, suggesting that the genomic organization of the AChR e subunit has been conserved throughout mammalian evolution (see table 1). Comparison of mouse and human intron sequences reveals an average homology of 54%, mostly resulting from blocks of identical sequence. Several potential regulatory elements in intronic regions were identified by computer analysis.34 By comparison with the corresponding mouse and rat sequences, we identified two conserved motifs of 10 bp containing a CANNTG consensus site in intron 3 and intron 8 (59-gccaggtggg-39 and 59gccacctgga-39, respectively). The nucleotide sequence of the protein-coding region is consistent with the previously published cDNA sequence of the AChR e subunit.5 The human amino acid sequence is identical with the mouse sequence. In addition, we found a polymorphism in exon 11 (e1233C/T) that does not result in the change of an amino acid. This polymorphism already has been described by Ohno et al. in an allelic frequency of 40/196.28 We identified e1233C/T in two of eight healthy controls from southern Germany. AChR e subunit–e1267delG in CMS patients. Mutation analysis of the AChR e subunit gene was carried out in 43 1566 NEUROLOGY 53 October (2 of 2) 1999
CMS patients. In 13 patients from 11 nonrelated families, we identified a homozygous deletion of a G corresponding to position 1267 (e1267delG) in exon 12 of the AChR e subunit gene. To exclude additional mutations, the complete coding region of the AChR e subunit gene was sequenced in patients harboring e1267delG. No other mutation was found. However, in exon 11, the polymorphism (e1233C/T) described earlier was identified homozygously in all patients harboring e1267delG. Where available, relatives were analyzed for e1267delG (figure). We found only affected siblings to be homozygous carriers of the mutation; all heterozygous carriers of e1267delG were asymptomatic (parents and siblings). Thus, heterozygousity for e1267delG is not sufficient to cause CMS, in
Figure. Restriction enzyme analysis in two patients diagnosed with congenital myasthenic syndromes (Patients 1 and 2), their asymptomatic parents (3, 4), and siblings (5, 6). A PCR fragment of 550 bp (549 bp in the mutated allele) containing exon 11 and exon 12 of the AChR e subunit was amplified. Restriction digest with XagI yields two fragments of 355 bp and 195 bp for the wild-type e subunit. The e1267delG mutation results in loss of the XagI site. Both the wild-type and mutant fragments are observed in the asymptomatic parents (3, 4) and the asymptomatic siblings (5, 6), indicating heterozygousity for e1267delG. Both patients (1, 2) are homozygous for e1267delG.
Table 2 Frequency of clinical features in 13 patients harboring e1267delG %
No. of patients affected
Ptosis
100
13/13
Weakness of bulbar muscles
100
13/13
Positive effect of AChE inhibitors
100
13/13
No worsening with age
100
13/13
Feature
Age at onset ,1 y
100
13/13
Ophthalmoparesis
92
12/13
EMG: decrement
78
7/9
Generalized weakness
77
10/13
Impaired ambulation
33
4/12
Delayed motor milestones
36
4/11
High arched palate
23
3/13
AChE 5 acetylcholinesterase; EMG 5 electromyography.
concordance with the autosomal recessive nature of the disease. Clinical phenotype of e1267delG patients. Of 11 nonrelated e1267delG families, 15 CMS patients were evaluated by questionnaires (table 2). In four families, two siblings each were affected. In two of these families, DNA samples and clinical data were not available for the affected sibling, thus data analysis was carried out in 13 affected individuals only. In addition, 11 unaffected siblings from six families were reported. Among the 11 CMS families, 7 are known to be of Gypsy ethnic origin. Of the seven Gypsy families, four originate from Hungary and still are living there. The other three Gypsy families are residing in Germany; two of them recently immigrated from Macedonia and Serbia. The third Gypsy family immigrated to Germany from Bohemia two generations ago. The four families who were not aware of a Gypsy ethnic origin immigrated to Germany from different southeast European countries, namely Macedonia (n 5 2), Turkey, and Kosovo. Consanguinity was not reported. At the time of the examination, the age of the patients varied from 6 months to 21 years; there were seven female and eight male patients. In all patients, first symptoms of CMS had occurred in early childhood, usually in the first months of life, with fluctuating ptosis, poor cry, and feeding difficulties. Respiratory failure in early childhood, precipitated by stress or infections, was reported in one patient only. Fatalities were not reported. In all patients, ptosis and bulbar weakness were present. Bulbar weakness was evidenced by difficulties in chewing or swallowing and involvement of facial muscles. Ophthalmoparesis was reported for 12 of the 13 patients, ranging from slight limitation of eye movements to marked ophthalmoplegia. Exercise-induced, generalized weakness of limb muscles, predominantly involving proximal muscles, was noted in most patients. Nevertheless, most patients were able to walk. In only four patients, ambulation clearly was restricted; two patients used wheelchairs for longer distances. The natural course of the disease was benign in all individuals, with little or no progression with age. Significant improvement of symptoms with age was reported in one patient. Remarkably, a positive response to anticholinesterase drugs was reported
in all patients. However, in some patients, residual myasthenic symptoms were refractory to long-time treatment with pyridostigmine (Mestinon, ranging from 1 to 8 mg/kg/ day). Repetitive stimulation of two distal and two proximal muscles was carried out in nine patients. Seven showed a pathologic decremental response in at least one of the stimulated muscles; two did not. Of the two patients with negative results for decremental response, one was examined by single-fiber electromyography and showed block of neuromuscular transmission; in the second patient, a 1-year-old baby, no further testing (single-fiber electromyography, facial muscles) was done. Genotype of e1267delG patients. To evaluate the hypothesis that the e1267delG mutation is caused by a founder effect in the southeastern European Gypsy population, genotype analysis was carried out using six polymorphic microsatellite markers on chromosome 17p13 close to the AChR e subunit gene. The marker closest to the gene, D17S1810 (genetic distance , 1 centiMorgan), showed strong linkage disequilibrium ( p , 0.0001) for an allele of 116 bp in e1267delG patients (20 of 22 chromosomes) compared with a control Gypsy population (3 of 18 chromosomes) and previously published Caucasian populations (Genome Database allele frequency: 2 of 27 chromosomes). All other markers examined in e1267delG patients showed a similar distribution of alleles as control Gypsy and control Caucasian populations (data not shown). These data provide strong genetic evidence that the e1267delG patients included in this study derive from a common ancestor (founder).
Discussion. The developmental, innervationdependent switch in the subunit structure of the AChR, where the g subunit is replaced by e, has focused much attention on these two subunits.2,3 Although the genomic sequences of the human a and g subunits were reported in 19834 and 1985,36 respectively, the complete genomic sequence and organization of the human AChR e subunit gene has not been published. The exon–intron arrangement of the e subunit gene, as determined by us, corresponds well to predictions based on the mouse AChR e subunit,33 dividing the protein-coding sequence into 12 exons by 11 introns. The relative sizes of mouse and human introns are similar, ranging from 82 to 1209 bp. Conserved motifs of 10 bp in intron 3 (59-gccaggtggg39) and intron 8 (59-gccacctgga-39) show 100% identity with corresponding mouse and rat sequences. Both motifs contain a CANNTG consensus site, also known as E-box, a putative target of myogenic factors such as MyoD. The relevance of this binding site and of other regulatory sequences for transcriptional regulation of the e subunit needs to be elucidated. The knowledge of intron sequences enabled us to analyze the complete coding region of the AChR e subunit in CMS patients. Several mutations of the AChR e gene causing autosomal recessive CMS have been reported.26-31 This indicates that mutations of the e subunit might be a frequent cause of this disorder. Indeed, in 13 patients from 11 nonrelated families, we identified a homozygous deletion of a G nucleotide in exon 12 of the e subunit gene (e1267delG). HeterozyOctober (2 of 2) 1999 NEUROLOGY 53 1567
gousity for e1267delG was found to be insufficient to cause CMS, in concordance with the autosomal recessive nature of the disease. e1267delG affects a region in the AChR e subunit that is prone to mutations. The deletion borders the intron 11– exon 12 boundary and leads to a frame shift after codon 422 with 63 missense codons followed by a stop codon. Consequently, e1267delG disrupts the cytoplasmic loop and the fourth transmembrane region (M4) of the e subunit. Alternatively, e1267delG may lead to aberrant splicing. Putative functional consequences on the expression of the AChR are either an incomplete assembly of the receptor subunits, a reduced expression of receptors containing the mutant e subunits, or a degradation of unstable receptors. In vitro expression studies of mutant AChRs recently were carried out by others.29 In this study, a single mutation of the AChR e subunit gene (e1267delG) was found in 13 (30%) of 43 CMS patients from 11 (31%) of 35 independent families. Middleton et al. identified e1267delG in three Greek and two Turkish CMS families.29 Croxen et al. identified e1267delG in five families originating from various geographic regions, including Pakistan, India, Egypt and Greece37 (Beeson, personal communication, 1998). In our study, all e1267delG families are of southeastern European or Gypsy ethnic origin. Epsilon1267delG was found in 50% (11/ 22) of CMS families of non-German ethnic origin compared with 0% (0/13) of German ethnic families. Clinical analysis of our patients revealed a phenotype for e1267delG that is similar to patients with other frame shifting or low-expressor missense mutations of the e subunit (see table 2). Like other CMS patients, e1267delG patients showed an early onset of disease and myasthenic symptoms involving bilateral ptosis, bulbar weakness, and a fatigable generalized weakness of leg and arm muscles. Moreover, all e1267delG patients showed a clear response to anticholinesterase medication, although some symptoms were refractory to long-term treatment. Most e1267delG patients showed a mild, nonprogressive course of CMS. A peculiar symptom in most e1267delG patients is ophthalmoparesis. In 30 CMS patients, in whom e1267delG was not detected, the frequency of ocular motility defects was about 50% only.38 Other clinical signs were analyzed to differentiate among e1267delG patients and other forms of CMS. In none of the e1267delG patients were cervical, scapular, or finger extensor muscles affected, as typically is seen in slow-channel CMS.32 Likewise, no repetitive muscle response to a single nerve stimulus was reported in electromyography of e1267delG patients. A high, arched palate was seen in some e1267delG patients. However, facial malformations, as previously described in Iranian CMS patients,39 were not described. Severity of disease is remarkably different among e1267delG patients, ranging from ptosis and involvement of extraocular muscles as the only symptom of CMS to disabling general weakness. To check for indicators of more severe phenotypes, 1568 NEUROLOGY 53 October (2 of 2) 1999
two patients, whose ambulation was severely restricted, were analyzed. Both patients, now 11 and 5 years old, respectively, showed a delay of motor milestones in infancy, which was found in 4 of 11 e1267delG patients only. In contrast, all patients showing normal motor development within the first 2 years of life had a mild CMS phenotype. Genotype analysis of our e1267delG families indicates that they derive from a common ancestor (founder) leading to CMS in the southeastern European Gypsy population. Similarly, a founder mutation in the g-sarcoglycan gene of Gypsies predating their migration from India was described recently.40 Our findings are of scientific as well as clinical relevance. CMS patients with an ethnic background, as described earlier, should undergo testing for the e1267delG mutation before performing other tests. We hypothesize that e1267delG may be found in a high proportion of these patients, making other tests unnecessary. Our findings support the notion that e1267delG is a frequent cause of CMS in patients of Gypsy ethnic origin, leading to a mild phenotype, further characterized by ophthalmoplegia and a good response to anticholinesterase drugs. Genetic analysis of CMS patients will reveal more genotype–phenotype correlations and hopefully will further facilitate genetic diagnosis, counseling, and therapy. Acknowledgment The authors thank Nancy Larochelle and Lefkos Middleton for helpful discussions and Nelly Lenz for technical assistance. The authors also thank David Beeson for communication of unpublished data.
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