Spectrum of Splicing Errors Caused by CHRNE Mutations Affecting Introns and Intron/Exon Boundaries K Ohno, a Tsujino, X-M Shen, M Milone, a G Engel
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1of5 ONLINE MUTATION REPORT J Med Genet: first published as 10.1136/jmg.2004.026682 on 1 August 2005. Downloaded from Spectrum of splicing errors caused by CHRNE mutations affecting introns and intron/exon boundaries K Ohno, A Tsujino, X-M Shen, M Milone, A G Engel ............................................................................................................................... J Med Genet 2005;42:e53 (http://www.jmedgenet.com/cgi/content/full/42/8/e53). doi: 10.1136/jmg.2004.026682 Patients Background: Mutations in CHRNE, the gene encoding the Patients 1–5 (respectively a 59 year old woman, a 23 year old muscle nicotinic acetylcholine receptor e subunit, cause man, a 2.5 year old girl, a 6 year old boy, and a 44 year old congenital myasthenic syndromes. Only three of the eight man) have moderate to severe myasthenic symptoms that intronic splice site mutations of CHRNE reported to date have have been present since birth or infancy, decremental EMG had their splicing consequences characterised. responses, and no AChR antibodies. All respond partially to Methods: We analysed four previously reported and five pyridostigmine. Patient 4 underwent an intercostal muscle novel splicing mutations in CHRNE by introducing the entire biopsy for diagnosis, which showed severe endplate AChR normal and mutant genomic CHRNEs into COS cells. deficiency (6% of normal) and compensatory expression of Results and conclusions: We found that short introns (82– the fetal c-AChR at the endplate. 109 nucleotides) favour intron retention, whereas medium to long introns (306–1210 nucleotides) flanking either or both Construction of CHRNE clones for splicing analysis sides of an exon favour exon skipping. Two mutations are of To examine the consequences of the identified splice site particular interest. Firstly, a GRT substitution at the 39 end of mutations, we used the previously constructed pRBG4- exon 8 predicts an R286M missense mutation, but instead CHRNE plasmid, which carries a cytomegalovirus (CMV) results in skipping of exon 8. In human genes, a mismatch of promoter and the entire CHRNE spanning 12 exons and 11 4 the last exonic nucleotide to U1 snRNP is frequently introns. For extended analysis of g.IVS10-9_c.1167dup16, compensated by a matching nucleotide at intron position we also constructed a pRBG4 minigene, spanning nucleotide 880 in exon 9 to nucleotide 1457 in exon 12, where position +6. CHRNE intron 8 has a mismatch at position +6, and +1 represents the first nucleotide of the first codon of the accordingly fails to compensate for the exonic mutation at mature peptide. position –1. Secondly, a 16 bp duplication, giving rise to two Mutations were engineered using the QuikChange Site 9 3 splice sites (g.IVS10-9_c.1167dup16), results in silencing Directed Mutagenesis kit (Stratagene). Presence of the of the downstream 39 splice site. This conforms to the expected mutation and absence of unwanted artefacts were scanning model of recognition of the 39 splice site, which confirmed by sequencing the entire insert. predicts that the first "ag" occurring after the branch point is http://jmg.bmj.com/ selected for splicing. Reverse transcription PCR analysis of cytoplasmic RNA of transfected COS cells Wild type and mutant CHRNE clones were introduced into COS- 7 cells, and cytoplasmic total RNA was isolated as described.4 olecular defects of presynaptic, synaptic, or post- We used two or more sets of PCR primers for each construct to synaptic proteins at the motor endplate impair screen for skipping of an exon and retention of an intron. neuromuscular transmission and result in congenital on September 27, 2021 by guest. Protected copyright. M 1 myasthenic syndromes (CMS). Mutations in the acetylcho- RESULTS line receptor (AChR) e subunit gene (CHRNE; OMIM 100725) Each patient carries two mutant CHRNE alleles cause endplate AChR deficiency and/or kinetic abnormalities We detected a total of seven CHRNE mutations in five of AChR. CHRNE mutations causing endplate AChR defi- patients (table 1). Patients 1, 2, and 3 carry homozygous ciency include 13 missense, 27 frameshift, 6 nonsense, 8 splice site mutations. Five mutations affect pre-mRNA splice site, 3 promoter region, and 1 chromosomal microdele- 1 splicing. Analysis of family members reveals that affected tion mutations. Only three of the eight splice site mutations siblings carry two mutant CHRNE alleles, whereas unaffected have had their consequences characterised: IVS7-2ARG2 and 3 relatives harbour one or no mutant allele (data not shown), IVS9+1GRT result in skipping of an adjacent exon, and indicating that each mutation is recessive. IVS9-1GRC causes retention of intron 9.4 Four of the five uncharacterised mutations were reported by us. Exclusive Splicing consequences of five novel and four expression of CHRNE by subsynaptic nuclei has previously previously reported mutations prevented analysis of splicing consequences of these muta- Muscle mRNA was available only from patient 4. In this tions. We recently reported that the cloned entire CHRNE patient, RT-PCR analysis revealed that 1259del23 causes exhibits the same splicing properties as its pre-mRNA in the retention of intron 11 (fig 1, table 1). As no muscle specimens native state in transfected COS cells.4 Therefore we used this were available from the other patients for mRNA analysis, we method to analyse the splicing consequences of four previ- introduced the four novel (table 1) and the four previously ously reported and five novel splice site mutations in CHRNE. reported (table 2) splice site mutations into the cloned entire CHRNE and analysed the cytoplasmic RNA of the transfected MATERIALS AND METHODS COS cells by RT-PCR. Unlike minigenes, entire CHRNE clones All human studies were in accord with the guidelines of the mostly yielded a single splicing product. RT-PCR analysis institutional review board of the Mayo Clinic. revealed that IVS4-2ARC, IVS6+1GRT, IVS10+2TRG, and www.jmedgenet.com 2of5 Ohno, Tsujino, Shen, et al J Med Genet: first published as 10.1136/jmg.2004.026682 on 1 August 2005. Downloaded from Table 1 Five splicing, one frameshift, and one missense 500 bp mutations in CHRNE in the five patients A IVS6 + 1G > T IVS7 + 2T > C 857G > T IVS10 + 2T > G c.1259_g.IVS11 + 15del23 Position on Splicing No. Mutation genomic DNA consequence IVS4 – 2A > C IVS6 – 1G > C IVS9 – 1G > A g.IVS10 – 9_c.1167dup16 1 g.IVS6-1GRC* 39 splice site of Active cryptic 39 Exon numbers and sizes (bp) intron 6 splice siteÀ 1 2 3 4 5 6 7 8 9 10 11 12 46 142 45 110 156 101 201 115 115 187 107 155 2 g.IVS9-1GRA* 39 splice site of Retention of Intron numbers and sizes (bp) intron 9 intron 9À 1 2 3 4 5 6 7 8 9 10 11 Skipping of 254 126123 129 306 334 82 1210 83 90 109 exon 10À B 3 g.IVS10-9_c.1167dup16* 16 bp Silencing of duplication downstream 39 comprising 8 bp splice siteÀ at 39 end of IVS4 – 2A > C: Skipping of exon 5 intron 10 and 8bpat59 end of exon 11 4 c.1259_g.IVS11+15del23 23 bp deletion Retention of IVS6 – 1G > T: Skipping of exon 6 comprising 8 bp intron 11` at 39 end of exon 11 and 15 bp at IVS6 – 1G > C: Activation of acryptic 3' splice site 59 end of intron 11 Intron 6 Exon 7 c.1033delG exon 10 (61st NA cagctgaccgtgcccccgtcccgcagAGAACGGCGAGTGG nucleotide) 5 c.857GRT (p.R286M) GRT substitution Skipping of c at 39 end of exon exon 8À 8 c.734CRT (p.P245L1) exon 7 (193rd NA IVS7 + 2T > C: Retention of intron 7 nucleotide) *Homozygous mutation. ÀCharacterised using transfected COS cells. `Characterised using muscle mRNA. NA, not applicable. 1We previously 857G > T: Skipping of exon 8 reported that P245L is a low expressor mutation that also prolongs Exon 8 Intron 8 channel opening events two fold.18 CTGAGCGTGCCGCTCCTGGGCAGgtgaagccggagcccccgcgggg T 857GRT cause skipping of an adjacent exon; IVS7+2TRC and 1259del23 result in retention of the mutant intron; IVS9- 1GRA causes both exon skipping and intron retention; IVS6- IVS9 – 1G > A: Retention of intron 9 and skipping of exon 10 1GRC activates a cryptic 39 splice site; and g.IVS10- 9_c.1167dup16 silences the downstream copy of the 39 splice site (fig 1, tables 1 and 2). IVS10 + 2T > G: Skipping of exon 10 http://jmg.bmj.com/ Why is the downstream copy of duplicated 39 splice ? sites silent g.IVS10 – 9_c.1167dup16: Silencing of downstream 3' splice site The 16 bp duplication (g.IVS10-9_c.1167dup16) generates Intron 10 Exon 11 Intron 10 Exon 11 two copies of 39 splice sites, but only the upstream copy is used for splicing. To understand the underlying mechanism, tcctaaggcccaccccggtgttttccccgccagCTGCCTTCcccgccagCTGCCTTCTG we engineered a series of artificial mutations into a minigene 16 bp depletion spanning CHRNE exons 9 to 12 (fig 2A and B). Firstly, we examined a role of the polypyrimidine tract of on September 27, 2021 by guest. Protected copyright. the upstream and downstream copies (fig 2C). The pyrimi- c.1259_g.IVS11 + 15del23: Retention of intron11 Exon 11 dine ratios in the polypyrimidine tract are the same for Intron 11 CGAGAGATCAGGAGGCCACCGGCGAGgtgggacaggagccagaggcgggtggagcgag the two copies (18/24 = 75% for the upstream copy and ^^^^^^^^^^^^^^^^^^^^^^^ 30/40 = 75% for the downstream copy), and are not likely to 23 bp depletion account for selection of the splice site. Substitution of ‘‘ac’’ for the invariant ‘‘ag’’ dinucleotide of the upstream copy Figure 1 Nine analysed CHRNE mutations affecting pre-mRNA activated the downstream copy (Mt-AC in fig 2D), indicating splicing. (A) The CHRNE gene structure is drawn to scale. Shaded areas indicate untranslated regions. Sizes of exons 1 and 12 represent those of that the increased distance from the branch point to the ‘‘ag’’ the coding regions. (B) Schematic presentation of identified splicing dinucleotide does not hinder splicing.