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Human Molecular Genetics, 2003, Vol. 12, No. 7 739-748
DOI: 10.1093/hmg/ddg089
© 2003 Oxford University Press

E-box mutations in the RAPSN promoter region in eight cases with congenital myasthenic syndrome

Kinji Ohno1,*, Menachem Sadeh2, Ilan Blatt2, Joan M. Brengman1 and Andrew G. Engel1

1Department of Neurology and Neuromuscular Research Laboratory, Mayo Clinic, Rochester, MN 55905, USA and 2Department of Neurology, Wolfson Medical Center, Holon, Israel

Received November 12, 2002; Revised January 23, 2003; Accepted February 1, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Myogenic determination factors are basic helix–loop–helix proteins that govern specification and differentiation of muscle cells, and bind to the E-box consensus sequence CANNTG in promoter regions of muscle-specific genes. No E-box mutation has been reported to date. RAPSN encodes rapsyn, a 43 kDa postsynaptic peripheral membrane protein that clusters the nicotinic acetylcholine receptor at the motor endplate. Transcriptional regulation mechanisms of RAPSN have not been studied. We here report two novel E-box mutations in the RAPSN promoter region in eight congenital myasthenic syndrome patients. Patient 1 carries -27C->G that changes an E-box at -27 to -22 from CAGCTG to GAGCTG. An allele harboring -27C->G is not transcribed in patient's muscle. Patients 2–8 are of Oriental Jewish stock of Iraqi or Iranian origin with facial malformations, and harbor -38A->G that changes another E-box at -40 to -35 from CAACTG to CAGCTG, which does not affect the consensus CANNTG sequence. Haplotype analysis shows that -38A->G arises from a common founder. For each mutation, position +1 represents the major transcriptional start site that we determine to be 172 nucleotides upstream of the translational start site. Electrophoretic mobility shift assays reveal that -38A->G gains, and -27C->G looses, binding affinity for different components of nuclear extracts of C2C12 myotubes. Luciferase reporter assays show that both -38A->G and -27C->G attenuate reporter gene expression in C2C12 myotubes, and that -27C->G additionally attenuates reporter gene expression in MyoD- or myogenin-transfected HEK cells. The -27C->G mutation also markedly attenuates the enhancer activity of an E-box on an SV40 promoter. Impaired transcriptional activities of the RAPSN promoter region predict reduced rapsyn expression and endplate acetylcholine receptor deficiency.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Myogenic determination factors (MDFs) are basic helix–loop–helix proteins that govern specification and differentiation of muscle cells (1). MDFs form heterodimers with ubiquitously expressed members of the E-protein family, E2/47, E2-2, E2-5, and HEB, and bind to the E-box consensus sequence CANNTG in promoter regions of muscle-specific genes. MDFs include four distinct proteins, MyoD (Myf3), myogenin (Myf4), Myf5, and Myf6 (Mrf4/herculin). MyoD and Myf5 are predominantly expressed in proliferating myoblasts prior to myotube differentiation, whereas myogenin and probably Myf6 are involved in terminal myotube differentiation (2). No E-box mutation in human has been reported to date.

RAPSN encodes rapsyn, a 43 kDa postsynaptic peripheral membrane protein that clusters the nicotinic acetylcholine receptor (AChR) in the postsynaptic membrane of the motor endplate (EP) (3). Rapsyn is an effector of neural agrin that is released from the nerve terminal to induce clustering of AChR at the EP (4). Targeted disruption of rapsyn impairs clustering of AChR at the EP (5). Rapsyn comprises distinct structural domains: a myristoylation signal at the N-terminus targets rapsyn to the membrane (6), seven tetratrico peptide repeats (TPRs) from codons 6–279 subserve rapsyn self-association (6,7), a coiled-coil domain at codons 363–403 interacts with the long cytoplasmic loop of each AChR subunit (8), a cysteine-rich RING-H2 domain at codons 363–402 interacts with the cytoplasmic domain of ß-dystroglycan (9), and there is a serine phosphorylation site at codon 406. Transcriptional activation mechanisms of RAPSN have not been studied to date.

Congenital myasthenic syndromes (CMS) are caused by molecular defects of presynaptic, synaptic or postsynaptic proteins. Mutations in the choline acetyltransferase gene (CHAT) reduce the catalytic efficiency or expression of the enzyme, and impair acetylcholine recycling at the motor nerve terminal (10). Mutations in the gene encoding the collagenic tail subunit (COLQ) of acetylcholinesterase (AChE) cause EP AChE deficiency (11,12). Mutations in AChR subunit genes alter AChR channel kinetics and/or cause EP AChR deficiency (13,14). EP AChR deficiency is caused mostly by mutations in the AChR {varepsilon} subunit gene (CHRNE), and rarely in other AChR subunit genes (15). We recently reported three RAPSN mutations (L14P in TPR1, N88K in TPR3, and 553ins5 in TPR5) in four patients with EP AChR deficiency (16). N88K was observed in six out of eight mutant alleles. None of the mutations prevents self-association of rapsyn, but each interrupts recruitment of AChR to rapsyn clusters. We here report two novel mutations in RAPSN E-boxes in eight CMS patients. Patient 1 (Pt 1) carries an E-box mutation and a previously characterized N88K mutation. Pts 2–8 are homozygous for another E-box mutation that arises from a common founder in the Near-East. The E-box mutations affect binding affinity for components of nuclear extracts of C2C12 myotubes, impair muscle-specific gene expression, and reveal critical E-boxes in the RAPSN promoter region.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Patients
All patients have had myasthenic symptoms since birth or infancy, negative tests for anti-AChR antibodies, and respond partially to anti-cholinesterase medications. All except Pts 3 and 5 had a decremental electromyogram (EMG) or an abnormal single fiber EMG study (Fig. 1).



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  		Figure 1. Pt 5 at rest (A) and trying to close his eyes (B). Front (C) and side (D) views of Pt 8. Pt 5 had surgical correction for bilateral ptosis, as well as for mandibular prognathism and malocclusion.

 
Pt 1, a 25-year-old North American woman, has mild to moderate fatigable weakness involving eyelid elevator, facial and limb-girdle muscles. Pts 2–8, all Jewish people from the Near East, range from 11 to 40 years of age; four are male and three are female. Parents of Pts 4, 5 and 7 are first cousins. Both parents of Pts 4, 5, 6 and 7 are of Iraqi origin; both parents of Pt 3 are of Iranian origin; Pt 4 was born to a father of Iranian and a mother of Iraqi origin; Pt 2 was born to a father of Turkish and a mother of Iraqi origin; both parents of Pt 8 originated from Yemen. Pts 2–8 are phenotypically similar in having mild to severe fatigable weakness of the masticatory muscles associated with mandibular prognathism, malocclusion, a high-arched palate, and crowded teeth. They also have moderate to severe fatigable eyelid ptosis without ophthalmoparesis, facial weakness and slurred or hypernasal speech. Cervical, trunkal and limb muscles are spared except in Pts 5 and 6, who have mild weakness of the cervical muscles, and Pt 7 who has slight weakness of the deltoid muscle. All patients have had a benign course. Clinical features of Pts 2–8 have previously been reported in detail (17).

Endplate studies in Pt 1
Cholinesterase staining of EPs on glutaraldehyde-fixed teased single muscle fiber of Pt 1 revealed that the EP regions were markedly dispersed (Fig. 2A and B). Ultrastructural analysis of the EPs showed shallow postsynaptic folds and clefts, few secondary clefts, and smaller than normal nerve terminals and postsynaptic regions (Fig. 2C), but the structural integrity of the pre- and postsynaptic regions was preserved. Morphometric analysis of 94 EP regions of 32 EPs showed that the mean nerve terminal and postsynaptic areas were reduced to 53 and 20% of normal, respectively.



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  		Figure 2. AChE-reactive EP regions in Pt 1 (A) and in a control subject (B). Note marked dispersion of EP regions over the fiber surface in the patient. (C) Ultrastructure of typical EP region in Pt 1. The postsynaptic region is shallow and displays only a few secondary clefts none of which communicates with the primary cleft in the plane of the section. Bar in A and B=25 µm; bar in C=1 µm.

 
Fluorescence microscopy studies of EPs demonstrated markedly reduced expression of rapsyn as well as AChR (Fig. 3D and H) compared to normal (Fig. 3B and F). The number of {alpha}-bungarotoxin binding sites per EP and the amplitudes of miniature EP potentials and currents were 24, 21 and 30% of the respective values observed in normal controls.



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  		Figure 3. Two-color fluorescence localization at EPs of AChE (A and C) with AChR (B and D), and of VAChT (E and G) with rapsyn (F and H), in a control (A, B, E, and F) and in patient 1 (C, D, G, and H). Note marked attenuation of the signal for AChR (D) and rapsyn (H) at the patient EP. Bar=50 µm.

 
Mutation analysis
We detected no mutations in the AChR {alpha}, ß, {delta} and {varepsilon} subunit genes in Pts 1 and 2. We next directly sequenced eight exons of RAPSN and their flanking untranslated regions, including the promoter region, and identified two mutant alleles in each patient. Pt 1 was heterozygous for N88K in TPR3 and -27C->G in the promoter region. Pts 2–8 were homozygous for -38A->G in the promoter region. We previously reported that N88K does not affect self-association of rapsyn, but hinders recruitment of AChR to rapsyn clusters (16). Neither -27C->G nor -38A->G was detected in 400 normal alleles.

The RAPSN promoter region harbors three consecutive putative E-boxes that conform to the CANNTG E-box consensus sequence (Fig. 4B). The -27C->G mutation changes the 3' E-box sequence from CAGCTG to GAGCTG. The -38A->G mutation changes the middle E-box sequence from CAACTG to CAGCTG, which renders the core dinucleotide of the middle E-box identical to that of the 3' E-box.



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  		Figure 4. (A) Transcriptional start sites of RAPSN. The major transcriptional start site (position +1, large arrow), observed in 26 out of 41 clones, is 172 nucleotides upstream of the translational start sites. Transcriptional start sites at -20, -11, -6, -5, -1, +7, +26, +37 and +61 (small arrows) were observed in 2, 1, 5, 2, 1, 1, 1, 1 and 1 clone, respectively. (B) Three putative E-boxes (CANNTG) and positions of identified mutations. Bars indicate wild-type (W) and mutant (M) fragments cloned into the pGL3-Promoter vector. The introduced mutations are shown by dots on the bars. For E2W, E2M, E3W and E3M, four copies of each element were inserted into the pGL3-Promoter vector. For E23W and E23M, a single copy was inserted into pGL3-Promoter. Single copies of E1W, E2W, E2M, E3W and E3M were also synthesized for EMSAs.

 
All patients also had four homozygous RAPSN polymorphisms: 456T/C causing no amino acid substitution, 1143T/C causing no amino acid substitution, as well as intronic IVS7+185G/A and IVS7+278A/G. The four polymorphisms were tightly linked to each other in 56 control alleles examined, and the allelic frequency of this polymorphic haplotype was 46/56 (82%). We used these polymorphisms in searching for a founder effect for -38A->G.

Transcriptional start sites
To relate the RAPSN mutations to the transcriptional start sites, we determined the transcriptional start sites of RAPSN using the RNA ligase-mediated rapid amplification of cDNA ends. We analyzed 41 clones and identified 10 different transcriptional start sites (Fig. 4A). The major transcriptional start site was 172 nucleotides upstream of the translational start site, and we designate the major transcriptional start site as position +1 in this report. Annotations of -27C->G and -38A->G are based on this observation.

Family analysis and founder effect
Family analysis revealed that affected members carry two mutant alleles, whereas unaffected members harbor no or one mutant allele (Fig. 5A and D). Therefore, each mutation is recessive. As no DNA was available from the father of Pt 1, we cloned a DNA segment spanning -27C->G and N88K from Pt 1. This revealed that -27C->G and N88K are heteroallelic. Allele-specific RT–PCR analysis of muscle mRNA of Pt 1 indicated that the allele harboring -27C->G is not transcribed (Fig. 5B).



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  		Figure 5. (A) Analysis of family members of Pt 1 (closed symbol and arrow). Unaffected members harbor no (open symbols) or one (half-shaded symbols) mutant allele. (B) Cloning of a PCR fragment spanning N88K and -27C->G reveals that the two mutations are heteroallelic in Pt 1 (data not shown). Allele-specific RT–PCR of muscle mRNA of Pt 1 shows that an allele carrying mutant -27G and wild-type N88 is not transcribed, whereas an allele with wild-type -27C and mutant K88 is transcribed in muscle. (C) Microsatellite markers (open circles) flanking RAPSN (closed circle) and haplotype analysis. Twelve haplotypes (I–XII) were observed in families 2–8 (F2–F8). PCR product sizes are indicated for each microsatellite marker. A physical map of the markers is drawn to scale according to the NCBI uniSTS database. Numbers in parentheses indicate approximate genetic distances from RAPSN in centiMorgan according to the deCODE recombination map (18). n.a., not available. (D) Haplotypes of family members. Propositi (arrows) and affected siblings (closed symbols) are homozygous for haplotype I, whereas unaffected members (half-shaded symbols) carry a single haplotype I, indicating that -38A->G arises from a common founder. Small symbols in family 5 indicate that no DNA was available.

 
As -38A->G was shared in Pts 2–8, we examined whether -38A->G arises from a common founder. To this end, we analyzed four single nucleotide polymorphisms within RAPSN and six microsatellite markers flanking RAPSN (Fig. 5C). Six microsatellite markers were within a ~3 Mbp segment according to the NCBI uniSTS database, or within a 0.85 cM stretch on the deCODE recombination map (18). We identified 12 haplotypes in families 2–8, and -38A->G was exclusively observed in haplotype I (Fig. 5D). Haplotype analysis confirmed that -38A->G arises from a common founder. In addition, divergent haplotypes observed in unaffected family members indicate that none of the families are closely related to each other.

Electrophoretic mobility shift assays
We employed electrophoretic mobility shift assays (EMSAs) to determine which of the three wild-type E-boxes (E1W, E2W and E3W in Fig. 4B) has muscle-specific binding properties. We found that E1W (complex 4) and E3W (complexes 4 and 5) confer muscle-specific binding, whereas E2W does not (Fig. 6). The three wild-type E-boxes also formed variable combinations of complexes 1, 2 and 3 with nuclear extracts of HEK cells; the bindings are likely to be due to ubiquitously expressed or fibroblast-specific E-box binding proteins.



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  		Figure 6. EMSAs of three wild-type E-boxes with nuclear extracts of HEK cells and C2C12 myotubes. E1W and E3W do, but E2W does not, show myotube-specific bindings. Complexes 1, 2 and 3 (dots) are formed by wild-type E-boxes in binding to nuclear extracts of HEK cells. Complex 4 (arrowhead) is formed by E1W and E3W in binding to nuclear extracts of C2C12 myotubes. Note that complex 4 moves slightly slower than complex 3 on a gel. Complex 5 (arrowhead) is exclusively formed by E3W in binding to nuclear extracts of C2C12 myotubes. Radio-labeled probes are indicated with asterisks.

 
We next examined the effects of -38A->G and -27C->G on EMSAs. As -38A->G changes the sequence of the middle E-box from CAACTG to CAGCTG, which thus acquires the sequence of the 3' E-box (Fig. 4B), we asked if -38A->G can also bind to muscle-specific proteins like E3W. Indeed, E2M carrying -38A->G formed complex 4 with nuclear extracts of C2C12 myotubes (lane 2 in Fig. 7A). Competition assay also demonstrated that the binding is specific to E2M (lanes 3–8 in Fig. 7A).



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  		Figure 7. (A) EMSAs of wild-type E2W and mutant E2M carrying -38A->G with nuclear extracts of C2C12 myotubes. E2M forms myotube-specific complex 4 (lane 2). E2M does, but E2W does not, compete for formation of complex 4 (lanes 3–8). (B) EMSAs of wild-type E3W and mutant E3M carrying -27C->G with nuclear extracts of C2C12 myotubes. E3W forms complexes 4 and 5 as in Figure 6, whereas E3M forms only complex 4. (C) Competition assays between wild-type E3W and mutant E3M using nuclear extracts of C2C12 myotubes. Both wild-type E3W and mutant E3M similarly compete for formation of complex 4, whereas wild-type E3W competes for formation of complex 5 more efficiently than mutant E3M. Complex numbers are the same as in Figure 6. Arrowheads point to myotube-specific complexes; dots point to a complex observed also in HEK cells.

 
We similarly analyzed the effect of -27C->G. Mutant E3M carrying -27C->G forms complex 4 like wild-type E3W, but no complex 5, in binding to nuclear extracts of C2C12 myotubes (Fig. 7B). Competition assays also demonstrated that complex 5 was more efficiently competed by wild-type E3W than mutant E3M, whereas complex 4 was similarly competed by wild-type E3W and mutant E3M (Fig. 6C).

To summarize, -38A->G gains binding affinity for formation of complex 4, whereas -27C->G loses binding affinity for formation of complex 5 in binding to nuclear extracts of C2C12 myotubes.

Transcription activities of wild-type and mutant RAPSN promoter region
To analyze the effects of the E-box mutations on RAPSN promoter activities, we first proved that the RAPSN promoter region spanning nucleotides -586 to +144 drives C2C12-myotube-specific gene expression (Fig. 8A). We also observed a moderate degree of promoter activity in C2C12 myoblasts, which is probably due to unsuppressed differentiation to myotubes. Alternatively, the RAPSN promoter may be partially active in myoblasts. Cotransfection of MyoD and myogenin also activated the RAPSN promoter region in HEK cells (Fig. 8A).



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  		Figure 8. (A) Promoter activities of wild-type and mutant RAPSN promoter regions. Compared to the pGL3-Basic construct, which harbors the firefly luciferase gene (Luc) without a promoter, wild-type RAPSN promoter region from -586 to +144 (shown by a line) drives the luciferase gene expression in C2C12-myoblasts and myotubes, as well as in MyoD- and myogenin-cotransfected HEK cells. The -38A->G mutation (closed circle) significantly reduces the reporter gene expression in C2C12 myoblasts/myotubes. The -27C->G mutation (closed circle) also significantly reduces the reporter gene expression in C2C12 myoblasts/myotubes, as well as in HEK cells cotransfected with MyoD and myogenin. *P<0.001 compared with the wild-type construct. Arrow points to the major transcriptional start site. (B) Enhancer activities of wild-type E2W and mutant E2M carrying -38A->G. Four contiguous copies of E2W and E2M fused to an SV40 promoter (Pro) of the pGL3-Promoter vector fail to enhance the reporter gene expression in the indicated cells. A single copy of the middle and 3' wild-type E-boxes (pGL3P-E23W) enhances an SV40 promoter. Introduction of -38A->G (closed circle in pGL3P-E23M) has no effects on the enhancer activity of E23W. (C) Enhancer activities of wild-type E3W and mutant E3M carrying -27C->G. Four contiguous copies of wild-type E3W fused to an SV40 promoter (Pro) activate reporter gene expression in MyoD- and myogenin-cotransfected HEK cells, as well as in C2C12 myotubes. The -27C->G mutation (closed circle in E3M) abolishes the enhancer effects. *P<0.001 compared with the wild-type E3W construct. Values indicate mean and SD in all the panels.

 
We next introduced -38A->G and -27C->G in the RAPSN promoter region and expressed the mutant constructs in HEK cells and C2C12 myocytes (Fig. 8A). We found that -38A->G significantly reduced the reporter gene expression in C2C12 myoblasts/myotubes. The -27C->G mutation significantly attenuated the reporter gene expression in C2C12 myoblasts and myotubes, as well as in MyoD- or myogenin-cotransfected HEK cells, but not in uncotransfected HEK cells.

Enhancer activities of wild-type and mutant RAPSN E-boxes
We next examined the effects of the E-box mutations on enhancement of an SV40 promoter by fusing four copies of E2W, E2M, E3W or E3M into pGL3-Promoter vector that carries an SV40 promoter and a firefly luciferase gene. We found that neither wild-type E2W nor mutant E2M carrying -38A->G enhances an SV40 promoter in MyoD- or myogenin-cotransfected HEK cells, or in C2C12 myotubes (Fig. 8B). We also observed that wild-type E23W, harboring both E2W and E3W, slightly enhanced an SV40 promoter (Fig. 8B). The -38A->G mutation, however, had no effects on this enhancement. The enhancement by E23W was similar to that of a single copy of E3W (data not shown), indicating that E2W exhibits no cooperation with E3W. Thus, neither wild-type E2W nor mutant E2M enhances an SV40 promoter alone or in collaboration with E3W.

The effect of -27C->G on enhancer activities was prominent. Four copies of wild-type E3W markedly enhanced reporter gene expressions in MyoD- and myogenin-cotransfected HEK cells, as well as in C2C12 myotubes. The -27C->G mutation abolished the enhancer effects (Fig. 8C).

To summarize, the wild-type RAPSN promoter region is active in MyoD- or myogenin-cotransfected HEK cells, and in C2C12 myoblasts/myotubes. The -38A->G mutation attenuates reporter gene expression in C2C12 myoblasts/myotubes. The -27C->G mutation reduces reporter gene expression in C2C12 myoblasts/myotubes, as well as in MyoD- or myogenin-cotransfected HEK cells. When enhancer activities of wild-type and mutant E-boxes are analyzed independent of the RAPSN promoter context, -38A->G does not affect SV40 promoter activities, whereas the -27C->G mutation markedly attenuates the enhancer activities.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We report two novel E-box mutations in the RAPSN promoter region in eight CMS patients. Impaired transcriptional activities of the RAPSN promoter region in myotubes predict reduced rapsyn expression and hence EP AChR deficiency. Indeed, RT–PCR analysis reveals that the mutant allele with -27G is not transcribed in muscle in Pt 1, and EP studies demonstrate deficiency of AChR and rapsyn in Pt 1.

Rapsyn is preferentially transcribed at the EP as are other EP-specific molecules (19–21). Cis-acting elements that confer synapse specific expression of RAPSN, however, have not been identified. In the AChR {delta} and {varepsilon} subunit genes, upregulation of gene transcription in subsynaptic nuclei is mediated by the Ets-binding site (CGGAA) (22,23), or the N-box (CCGGAA) (24,25). The Ets-binding site also drives synapse-specific gene expression of utrophin (26,27), AChE (28,29), and probably also the expressions of the AChR {alpha} subunit and ß2 laminin (30). On the other hand, synaptic expression of rapsyn is not affected by suppression of the Ets transactivating factor in transgenic mice, indicating that synaptic transcription of RAPSN is probably regulated by an Ets-independent mechanism (30).

In AChR subunit genes, an E-box drives muscle-specific gene expression (31,32), and also governs electrical activity-dependent repression of gene expression in extrasynaptic myonuclei (33,34). As most muscle genes harboring an E-box are not regulated by electrical activity, other regulatory elements must cooperate with the E-box for electrical activity-dependent transcriptional repression of the AChR subunit gene expression. The cooperating elements include the SV40 core enhancer-like element and SP1 in the rat {delta} subunit (35), and AR-160 and AR-120 in the chicken {alpha} subunit (36). We found that the middle and 3' E-boxes of RAPSN regulate muscle-specific gene expression, but we do not know if the identified E-boxes also mediate downregulation of extrasynaptic gene expression.

Denervation of skeletal muscle upregulates transcription of RAPSN 2–3-fold, whereas transcription of AChR {alpha} subunit gene is increased 30–500-fold (37–39), which is probably driven by increased MDFs after denervation (39,40). A low degree of MDF-responsiveness of RAPSN in denervated muscle may indicate that MDFs are not major transactivating factors for RAPSN, or an unidentified cis-factor suppresses MDF-responsiveness of RAPSN in denervated muscle.

The -27C->G mutation changes the 3' E-box from CAGCTG to GAGCTG. Crystallography of MyoD-DNA binding complex reveals that glutamate at position 118 (E118) of MyoD forms hydrogen bonds with the CA dinucleotides of CANNTG (41). Arginine at 111 (R111) of MyoD also forms a hydrogen bond with the last G of CANNTG on the opposite strand, and the conformation of R111 is essential for transcriptional activation of MDFs (42). As E118 and R111 are conserved in MyoD, myogenin, Myf5, and Myf6, replacement of this essential C:G pairing to G:C by -27C->G probably accounts for loss of binding affinity for complex 5 in EMSA, as well as loss of the transcriptional activity of the RAPSN promoter region (Fig. 8A) and of the enhancer activity of the 3' E-box (Fig. 8C).

The -38A->G mutation converts the middle E-box of CAACTG to CAGCTG. Binding site selection studies reveal that the preferred binding sequence for MyoD homodimer is CAGCTG (43). Crystallographic analysis also demonstrates that arginine at position 117 (R117) and leucine at 122 (L122) of MyoD make GC the preferred dinucleotides at the central core position of CANNTG (41). As R117 and L122 are conserved in MyoD, myogenin, Myf5 and Myf6, -38A->G may confer binding to MDFs. Indeed, EMSAs demonstrate that -38A->G gains binding affinity for a nuclear component of C2C12 myotubes (complex 4 in Fig. 7A), as we observe in the 5' and 3' E-boxes. It is interesting to note that a single nucleotide substitution within non-conserved core dinucleotides of CANNTG alters binding affinity for a DNA binding molecule.

Luciferase reporter assays reveal that -38A->G compromises transcription activation in the context of the RAPSN promoter region (Fig. 8A). The wild-type middle E-box, however, does not enhance an SV40 promoter, and neither does -38A->G (Fig. 8B). As cooperative transcriptional regulation of an E-box with another E-box or with other cis-acting elements is observed in creatine kinase (44), desmin (45), AChR {alpha} subunit (31), AChR ß subunit (46), MyoD1 (47), and skeletal muscle sodium channel NaV1.4 (48,49), we searched for cooperative effect of the middle E-box on the 3' E-box, and found the middle E-box has no effect on the enhancer activities of the 3' E-box (Fig. 8B). The middle E-box probably cooperates with other unidentified cis-acting element(s), including the 5' E-box, located between -586 and +144 in the RAPSN promoter region, and -38A->G affects the collaboration. Further molecular dissection of the RAPSN promoter region will reveal the specific cis-acting element(s) that cooperate with the middle E-box.

Pts 2–8 and their affected siblings share unusual facial malformations and carry the common -38A->G mutation. As most transcription factors are developmentally regulated, these patients probably had severe rapsyn as well as AChR deficiency during embryogenesis, which produced the facial malformations. Alternatively, as haplotype analysis revealed a founder effect for -38A->G, a defective gene closely linked to RAPSN may account for the facial malformations, although no genes close to RAPSN are known to be associated with facial malformations (NCBI Human Map Viewer, Build 30).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Muscle specimens
Intercostal muscle specimens were obtained intact from origin to insertion from Pt 1, and from control subjects without muscle disease undergoing thoracic surgery. All human studies were in accord with the guidelines of the Institutional Review Board of the Mayo Clinic. EPs were localized for electron microscopy and analyzed by established methods (50,51).

AChR and AChE were colocalized in cryostat sections with rhodamine-labeled {alpha}-bungarotoxin and a monoclonal anti-AChE antibody (52). The vesicular ACh transporter (VAChT) was colocalized with a mouse-anti-rapsyn monoclonal (mAb 1234, 2 µg/ml; gift of Stanley Froehner) and polyclonal rabbit anti-VAChT (1/500; gift of J.D. Erickson) as primary antibodies, and with fluorescein-isothiocyanate (FITC)-labeled donkey anti-mouse IgG (5 µg/ml) and CY3-labeled donkey anti-rabbit IgG (1 µg/ml) as second antibodies (Jackson Laboratories).

The number of AChRs per EP was measured with [125I]{alpha}-bungarotoxin (53). Recordings of miniature EP potentials and currents were performed as described elsewhere (53,54).

Sequencing procedures
After PCR amplification in 25 µl, we eliminated unincorporated dNTPs and primers by adding 5 units of shrimp alkaline phosphatase (Amersham USB) and 25 units of exonuclease I (Amersham USB), followed by incubation at 37°C for 15 min and enzyme inactivation at 80°C for 15 min (55). Plasmids were purified by the QIAprep Spin Miniprep Kit (Qiagen). PCR products and plasmids were sequenced with an ABI 377 DNA sequencer (Applied Biosystems) using fluorescently labeled dideoxy terminators.

Mutation analysis
DNA was isolated from muscle and blood as described (56). We directly sequenced all exons with their flanking noncoding regions of the AChR {alpha}, ß, {delta} and {varepsilon} subunit genes (56) and of RAPSN (16). For RAPSN, we sequenced ~800 bp upstream of the translational start site, and 150 bp or more of the flanking introns.

We employed allele-specific PCR to screen 400 normal alleles for -38A->G and -27C->G. We tracked mutations in family members by direct sequencing (-38A->G), allele-specific PCR (N88K), or PvuII restriction analysis (-27C->G). For Pt 1, we PCR-amplified a RAPSN fragment flanking -27C->G and N88K from DNA of Pt 1, and cloned it into a pGEM-T vector (Promega). To determine whether -27C->G and N88K were heteroallelic or homoallelic, we isolated and sequenced eight pGEM-T clones.

Allele-specific RT–PCR of muscle in Pt 1
To examine if an allele carrying -27C->G is transcribed in muscle of Pt 1, we performed allele-specific RT–PCR. We first isolated mRNA from EP-containing muscle specimen by Micro-FastTrack mRNA Isolation Kit (Invitrogen), treated mRNA with 2.8 units of DNase, and then synthesized cDNA using Superscript II (Invitrogen) and random primers (Invitrogen) according to the manufacturer's recommendations. For allele-specific RT–PCR, the wild-type N88-allele-specific forward primer was 5'-CCTCCTGGAGAGCTACCTGcAC-3' and the mutant K88-specific forward primer was 5'-CCTCCTGGAGAGCTACCTGcAA-3'. A lower case ‘c’ at position -3 represents an artificial mismatch to enhance discrimination of the two alleles (57). The reverse primer was 5'-TTCTGGAAGACGCTGAGG-3'. RT–PCR would yield a 180 bp fragment from each specific allele.

Haplotype analysis
To test for a founder effect in families harboring -38A->G, we analyzed four single nucleotide polymorphisms within RAPSN by direct sequencing, and six microsatellite markers flanking RAPSN (Fig. 5C) by the GeneScan Analysis, both using an ABI 377 automated DNA sequencer (Applied Biosystems). For microsatellite analysis, we synthesized PCR primers according to the Genome Database (http://www.gdb.org/), and attached a 6-FAM fluorescent dye to the 5' end of forward primers using an ABI 394 DNA Synthesizer (Applied Biosystems).

Identification of transcriptional start sites
We isolated mRNA from normal EP-containing skeletal muscle using the Micro-FastTrack mRNA isolation kit (Invitrogen). We then determined the transcriptional start sites of RAPSN using the RNA ligase-mediated rapid amplification of cDNA ends (GeneRacer Kit, Invitrogen). Briefly, 5' phosphate of truncated or degraded mRNA was removed by calf intestinal phosphatase. The cap structure of full-length mRNA was then removed by tobacco acid pyrophosphatase, which does not remove the 5' phosphate of mRNA. An RNA oligonucleotide was ligated to the 5' end of full-length mRNA using T4 RNA ligase. After synthesizing cDNA with AMV reverse transcriptase using random primers, we performed nested RACE (rapid amplification of cDNA ends) RT–PCR. Forward primers for the first-round and nested PCR were supplied with the kit and are located within the ligated RNA oligonucleotide. The first-round reverse primer was 5'-TTCTGGAAGACGCTGAGG-3' at 577–594, where nucleotide +1 represents the major transcriptional start site (see Results). The nested reverse primer was 5'-TCTGTCTGGTTGGACTGGTACA-3' at 219–240. The RT–PCR products were then ligated into pCR4 vector. We isolated 41 clones carrying inserts and determined the transcriptional start sites by sequencing their inserts.

Cloning of myogenic determination factors
The human MYOD1 cDNA encoding MyoD (I.M.A.G.E. clone ID 2961494) and the human MYOG cDNA encoding myogenin (I.M.A.G.E. clone ID 4106815) were obtained from ATCC. Using the I.M.A.G.E. clones, we PCR-amplified the entire coding regions of MYOD1 (nucleotides 1–963) and MYOG (nucleotides 1–675), where position +1 represents a translational start site. PCR primers introduced an XbaI site and a Kozak consensus sequence (5'-CCACC-3') (58) at the 5' end, and an EcoRI site at the 3' end of the PCR product. The PCR products were then ligated into a CMV-based mammalian expression vector pRBG4 (56). Absence of PCR artifact was confirmed by sequencing the entire inserts.

Preparation of nuclear extracts for EMSAs
HEK and C2C12 cells were grown to confluency in a 150 cm2 flask. C2C12 myoblasts were induced to differentiate in DMEM supplemented with 2% horse serum (Invitrogen) and 10 µg/ml insulin (Invitrogen) for 2 days. Cells were washed twice with 10 ml of cold PBS containing 5 mM EDTA, centrifuged at 1000g for 10 min at 4°C, and resuspended in 1 ml of buffer A (10 mM HEPES, pH 7.8, 1.5 mM MgCl2, 10 mM KCl, 1 mM DTT, 5 µg/ml leupeptin, 1 mM PMSF, and 0.1 mM benzamidine). Cytoplasmic membranes were disrupted by passing cells through a 27-gage needle on a 1 ml syringe about six times. Nuclei were centrifuged at 16 000g for 15 s at 4°C, and the nuclear pellet was resuspended in 3 vols of buffer B (20 mM HEPES, pH 7.8, 25% glycerol, 420 mM KCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, and protease inhibitors). After incubation on ice for 20 min, the nuclei were passed through a 27-gage needle on a 1 ml syringe about six times. The debris was removed by centrifugation at 16 000g for 15 s at 4°C. Protein concentration was determined by the BCA method (Pierce), and the nuclear extracts were stored at -70°C in aliquots.

EMSAs
We synthesized five pairs of complementary oligonucleotides, E1W, E2W, E2M, E3W and E3M (Fig. 4B). As the end-labeling efficiency of a C nucleotide is lower than other nucleotides (59), we included one or two more nucleotides when a C nucleotide is present at the 5' end.

Three picomoles of a single-stranded oligonucleotide were end-labeled using T4 polynucleotide kinase (Roche) and [{gamma}-32P]ATP (Amersham Biosciences). Unincorporated nucleotides were eliminated using a Microspin G-25 column (Amersham Biosciences). Complementary strands were annealed in 100 µl annealing buffer (10 mM Tris–HCl, pH 8.0, 50 mM NaCl, and 1 mM EDTA) at 99°C for 3 min followed by slow cooling to room temperature. Competitor oligonucleotides were similarly annealed at variable concentrations but without 5' end labeling. Two microliters of nuclear protein extract were incubated at room temperature for 10 min in 20 mM HEPES, pH 7.8, 50 mM KCl, 3 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol, 10% glycerol, and 0.5 µg poly (dI-dC)cpoly (dI-dC) (Sigma) in 10 µl, with or without variable amounts (0.3–4.5 pmol) of competitor DNA. Labeled DNA probe (0.03 pmol) was added to the reaction mixture and incubated for 20 min at room temperature. The reaction mixtures were loaded onto a 6% Novex DNA Retardation Gel (Invitrogen). The gel was run at 4°C in 44.5 mM Tris, 44.5 mM boric acid, and 1 mM EDTA at 100 V for 80 min, dried, and imaged by the Molecular Imager FX (Bio-Rad). The band intensities were quantified using NIH Image 1.61.

Construction of luciferase reporter vectors
We constructed luciferase reporter vectors to measure the promoter activities of wild-type and mutant human RAPSN promoter regions in HEK fibroblasts and in C2C12 myoblasts/myotubes. Using control human genomic DNA, we PCR-amplified the RAPSN promoter region spanning nucleotides -586 to +144, where position +1 represents the major transcriptional start site, and inserted it into KpnI–HindIII sites of pGL3-Basic vector (Promega) that carries a firefly luciferase gene. For KpnI, we used a native restriction site at -586 to -581 of RAPSN. For HindIII, we introduced a restriction site at the 5' end of the PCR primer. KpnI and HindIII treatment of pGL3-Basic eliminated a noncanonical E-box at the multiple cloning site of the vector (60). The -38A->G and -27C->G mutations were introduced to the wild-type RAPSN construct using the QuikChange site directed mutagenesis kit (Stratagene). Absence of PCR artifacts was confirmed by sequencing the entire inserts for each construct.

To analyze enhancer activities of wild-type and mutant E-boxes, we introduced four contiguous copies of each E-box into the pGL3-Promoter vector (Promega) that carries an SV40 promoter and a firefly luciferase gene. We synthesized complementary wild-type (W) or mutant (M) oligonucleotides harboring four contiguous E-box copies of E2W, E2M, E3W and E3M, or a single copy of E23W and E23M (Fig. 4B) with linker nucleotides for KpnI at the 5' end and XmaI at the 3' end. We phosphorylated the 5' end of synthesized oligonucleotides with T4 polynucleotide kinase (Roche), diluted the oligonucleotides to 10 µM, and then annealed complementary oligonucleotides by heating for 2 min at 95°C and slowly cooling down to room temperature. The phosphorylated double-stranded oligonucleotides were then inserted into the pGL3-Promoter vector.

Luciferase reporter assays
HEK fibroblasts and C2C12 myoblasts were obtained from ATCC and were cultured in HEPES-buffered DMEM supplemented with 10 and 20% fetal bovine serum (Invitrogen), respectively. Approximately 1.5x105 HEK fibroblasts and 0.5x105 C2C12 myoblasts were seeded into a six-well plate 24 h before transfection. Cells were transfected with 1 µg of each firefly luciferase reporter plasmid (pGL3-Basic or pGL3-Promoter vector) and 50 ng of phRL-TK (Promega) that carries a humanized Renilla luciferase gene driven by the herpes simplex virus thymidine kinase promoter, with or without 50 ng of pRBG4-MYOD1 or pRBG4-MYOG, using 3 µl of FuGENE 6 transfection reagent (Roche) according to the manufacturer's recommendations. Sixteen hours later, we added fresh DMEM supplemented with 10% (for HEK cells) or 20% (for C2C12 myoblasts) fetal bovine serum. To induce myotube differentiation from C2C12 myoblasts, we changed to DMEM with 2% horse serum (Invitrogen) and having 10 µg/ml insulin (Invitrogen). Proteins were extracted 48 hrs after transfection using 500 µl of Passive Lysis Buffer (Promega). Firefly and Renilla luciferase activities were determined using the Dual Luciferase Reporter Assay kit (Promega) in a Turner Design Luminometer (TD 20e). Each experiment was done in triplicate. Relative luciferase activity unit (RLU) is defined as the firefly luciferase activity divided by the Renilla luciferase activity.

In the course of luciferase assays, we noticed that myogenin enhanced expression of internal control phRL-TK vector 5-fold, and MyoD enhanced it 2-fold. Mutagenesis studies of phRL-TK, as well as absence of similar enhancement in CMV promoter-based pRL-CMV and in SV40 promoter-based pRL-SV40, revealed that CAGCTG at positions 529–534 (accession number AF362545) of phRL-TK within the TK promoter region accounts for this enhancement (data not shown). We therefore normalized the firefly luciferase activities by activities observed in empty pGL3-Basic or empty pGL3-Promoter for all the luciferase assays.


   ACKNOWLEDGEMENTS
 
This work was supported by the National Institutes of Health Grants NS6277 and an MDA research grant to A.G.E.


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +1 5072845102; Fax: +1 5072845831; Email: ohnok{at}mayo.edu Back


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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