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A newly identified chromosomal microdeletion of the rapsyn gene causes a congenital myasthenic syndrome
Neuromuscular Disorders 14 (2004) 744–749 www.elsevier.com/locate/nmd
Case report
A newly identified chromosomal microdeletion of the rapsyn gene causes a congenital myasthenic syndrome Juliane S. Mu¨llera,1, Angela Abichta,1, Hans-Ju¨rgen Christenb, Rolf Stuckaa, Ulrike Scharac, Wilhelm Mortierd, Angela Huebnere, Hanns Lochmu¨llera,* a
Department of Neurology and Gene Center, Friedrich-Baur-Institute, Ludwig-Maximilians-University, Munich, Germany b Department of Neuropediatrics, Kinderkrankenhaus auf der Bult, Hannover, Germany c Department of Neuropediatrics, Staedtische Kliniken, Neuss, Germany d Department of Pediatrics and Pediatric Neurology, Ruhr-University of Bochum, Bochum, Germany e Department of Pediatrics, Technical University Dresden, Dresden, Germany Received 29 January 2004; received in revised form 1 June 2004; accepted 9 June 2004
Abstract The objective is mutation analysis of the RAPSN gene in a patient with sporadic congenital myasthenic syndrome (CMS). Mutations in various genes encoding proteins expressed at the neuromuscular junction may cause CMS. Most mutations affect the epsilon subunit gene of the acetylcholine receptor (AChR) leading to endplate AChR deficiency. Recently, mutations in the RAPSN gene have been identified in several CMS patients with AChR deficiency. In most patients, RAPSN N88K was identified, either homozygously or heteroallelic to a second missense mutation. A sporadic CMS patient from Germany was analyzed for RAPSN mutations by RFLP, long-range PCR and sequence analysis. Clinically, the patient presents with an early onset CMS, associated with arthrogryposis multiplex congenita, recurrent episodes of respiratory insufficiency provoked by infections, and a moderate general weakness, responsive to anticholinesterase treatment. The mutation RAPSN N88K was found heterozygously to a large deletion of about 4.5 kb disrupting the RAPSN gene. Interestingly, an Alu-mediated unequal homologous recombination may have caused the deletion. We hypothesize that numerous interspersed Alu elements may predispose the RAPSN locus for genetic rearrangements. q 2004 Elsevier B.V. All rights reserved. Keywords: Congenital myasthenic syndrome; Rapsyn; Mutation N88K; Chromosomal deletion; Neuromuscular junction
1. Introduction Congenital myasthenic syndromes (CMS) are a heterogeneous group of disorders, where neuromuscular transmission is impaired by different inherited defects [1]. Molecular genetic studies led to the identification of numerous pathogenic mutations in various genes, encoding presynaptic (choline acetyltransferase, ChAT) [2–5], synaptic (collagenic tail subunit of acetylcholinesterase, ColQ) [6–11], and postsynaptic proteins (subunits of acetylcholine
* Corresponding author. Address: Genzentrum Mu¨nchen, Feodor-LynenStr. 25, 81377 Mu¨nchen, Germany. Tel.: C49-89-2180-76887; fax: C4989-2180-76999. E-mail address: [email protected] (H. Lochmu¨ller). 1 These authors contributed equally to this work. 0960-8966/$ - see front matter q 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2004.06.010
receptor, AChR and rapsyn) [12]. The majority of CMS affect postsynaptic functions. Mutations in AChR subunit genes alter AChR channel kinetics and/or cause endplate (EP-) AChR deficiency [12]. Especially mutations in the gene CHRNE encoding the 3 subunit of the AChR are responsible for CMS with EP-AChR deficiency [13,14]. In addition, mutations in the RAPSN gene have been shown to cause postsynaptic CMS with EP-AChR deficiency [15–21]. Rapsyn (acetylcholine receptor-associated protein of the synapse), a peripheral membrane protein of skeletal muscle is essential for clustering nicotinic acetylcholine receptors at high density in the postsynaptic membrane. In the great majority of CMS patients with underlying RAPSN mutations, a specific missense mutation of the gene (RAPSN N88K) has been identified homozygously or compound heterozygously to a second point mutation
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(missense, frame-shift, stop, splice-site or promoter mutations). Here we report the first chromosomal microdeletion of the RAPSN gene.
2. Material and methods Patients, DNA samples. Venous blood samples were obtained from the CMS patient as well as from his unaffected brother and unaffected parents. All studies were carried out with informed consent of the patient’s parents and were approved by the institutional ethics review board. Genomic DNA was isolated using a blood and tissue culture DNA extraction kit according to the manufacturer’s recommendations (Wizard Genomic DNA Purification Kit, Promega, Mannheim, Germany). Sequence analysis, RFLPs. PCR primers were designed based on the published genomic structure [GenBank accession number AC090559/gi:22002211, mRNA AF449218/gi19310212] of the RAPSN gene. Nucleotide positions are given according to the genome assembly July 2003 of the University of California Santa Cruz Genome Bioinformatics (http://genome.ucsc.edu). The Repeat Masker program provided by this website was used to analyze the nature of the sequences flanking the deletion breakpoints. In the patient, all eight exons, flanking intronic regions, and the promoter of the RAPSN gene were amplified by PCR and sequenced. PCR-amplified fragments were purified with the NucleoSpin Extract kit (Macherey– Nagel, Du¨ren, Germany) and sequenced with an Applied Biosystems model 3100 Avant DNA sequencer and fluorescein-labeled dideoxy terminators (Perkin–Elmer, Foster City, CA, USA). Screening for the mutation N88K (264COA) in exon 2 of the RAPSN gene was performed as described previously [18]. A 118 bp fragment in exon 2 was amplified by PCR using primers 5 0 -gaggatgccgacttcctcctgg3 0 and 5 0 -tggtaccaggcagcccaaggc-3 0 . The mutation N88K (264COA) creates a new BseNI site. In addition, all 12 exons, adjacent intronic regions and the promotor region of the AChR 3-subunit gene [CHRNE, GenBank accession number AF105999/gi4580858] were amplified by PCR and sequenced to exclude a CHRNE mutation in the patient. Genotype and polymorphism analysis. For haplotype analysis five polymorphic microsatellite markers on
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chromosome 11p11.2 flanking the RAPSN gene (D11S986, D11S1344, D11S4109, D11S4076, D11S1883) were chosen based on information obtained from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) and the University of California Santa Cruz Genome Bioinformatics (http://genome.ucsc.edu). Markers were amplified by PCR using fluorescence-labeled 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, USA). Determination of the deletion breakpoints. The longrange polymerase chain reaction was carried out on genomic DNA of the patient and his family using the Expand Long Template PCR System from Roche (Basel, Switzerland). A panel of specific primers in sense and antisense direction was used to determine the deletion breakpoints (Table 1).
3. Results Clinical data. The German patient, currently 3 years of age, is the second child of non-consanguineous healthy parents. His 17-months-old brother is healthy. The index patient was born after a 36-week pregnancy. The mother noted decreased fetal movements. The spontaneous vaginal delivery was complicated by a breech presentation. Postpartum, multiple hematomas and an arthrogryposis multiplex congenita, with multiple joint contractures of upper and lower limbs were reported. At day 3 postpartum, assisted ventilation was required for a few days. Since birth, severe difficulties in feeding and sucking were observed that prevented breast-feeding. Moreover, an open mouth and intermittent strabism were noted. Repeatedly, viral infections of the upper airways led to abnormal fatigue, weakness and respiratory insufficiency that required hospitalization. At the age of 15 months, a daytime-dependent eyelid ptosis was observed. Whereas cognitive development was normal, motor milestones were moderately delayed. Overall, only a slow and gradual improvement of the general condition occurred until a neuromuscular transmission defect was diagnosed and specific therapy was initiated. The patient was diagnosed with CMS at the age of 20 months, when a respiratory infection was accompanied
Table 1 Oligonucleotide sequences Name
Sequence
Position
RP forward R2 forward R2 reverse R6 forward AluSx forward 3 0 -UTR reverse L1CM4 reverse rs4282946 reverse
5 0 -TTGCCCTGGGGCAGGAAGAAGC 5 0 -AGGCTGGGGTCCAAGGCTCAGAGT 5 0 -GCCACAGGGTGTGTGCCTCA 5 0 -ACCCTGTGCTTCCCTGTGAGCA 5 0 -ACCAACATGGTGAAACTCT 5 0 -CCACCCACTGCACGTCAGCTTC 5 0 -TGAAATCCATGAGTTCACA 5 0 -GAGCCCATCCAAGCTGGCTCC
RAPSN promoter region RAPSN intron 1 RAPSN intron 2 RAPSN intron 5 RAPSN intron 6 Telomeric to RAPSN 3 0 -UTR Between rs4282946 and L1CM4 telomeric to SNP rs4282946
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by sudden development of severe respiratory insufficiency requiring assisted ventilation for 1 week. At the age of 21 months, the general physical and neurological examination revealed mild bilateral ptosis with mild restriction of extraocular movements in all directions of gaze. Bulbar weakness was apparent by a weak cough and involvement of facial muscles. In addition, the face appeared slightly elongated. The patient’s mouth was opened and the palate appeared to be high arched. Moderate contractures of knee and elbow joints were noted. Exercise-induced, generalized weakness of limb muscles, predominantly involving proximal muscles, was also noted. The child was able to stand with assistance, but was not yet able to walk unassisted. The anti-AChR antibody test was negative. Repetitive stimulation (3/s) of two distal (right N. medianus and right N. ulnaris) as well as two proximal motor nerves (right and left N. accessorius) revealed no decremental response of the compound muscle action potential at rest. An intravenous edrophonium (CamsilonR) test was unequivocally positive. A therapy with pyridostigmine bromide (MestinonR) at a moderate dosage (2.4 mg/kg per day) was started and resulted in immediate and significant clinical improvement of ptosis, motor skills and behavior. In particular, the child was able to walk independently only one day after treatment was initiated. His subsequent clinical course has been stable, with further improvements in motor development. Recurrent infections were successfully managed by intermittent increase of the pyridostigmine dosage or by decrease of the dosing intervals, respectively. Moreover, a slow, but steady regression of the contractures was seen. Mutational analysis. Sequence analysis of the coding and the promoter region of the RAPSN gene revealed a heterozygous C-to-A transition in exon 2 at nucleotide position 264 (264COA) in the index patient II/2 of our German CMS pedigree. The mutation leads to an amino acid exchange (N88K) that has previously been described by us and others as a recessive CMS mutation. Restriction analysis (Fig. 1A) revealed that unaffected family members are either carrying the mutation N88K heterozygously (I/1 and II/1) or do not carry the mutation (I/2). This is in accordance with an autosomal-recessive trait. However, no additional mutation was found by PCR-based sequence analysis of the coding and promoter region of the patient’s RAPSN gene. Therefore, we hypothesized that a chromosomal deletion or a chromosomal rearrangement of the maternal RAPSN allele may have been missed by our analysis. Identification and characterization of the deletion. Haplotype analysis using five polymorphic microsatellite markers on chromosome 11p11.2 flanking the RAPSN gene (D11S986, D11S1344, D11S4109, D11S4076, D11S1883) excluded an extensive chromosomal rearrangement. In addition, maternity of the patient’s mother was verified since the patient and his mother share the same allele (data not shown). Haplotype analysis confirmed that the index patient and his unaffected brother both inherited the N88K allele from their father.
Fig. 1. (A) Restriction enzyme analysis in the CMS family. To detect the RAPSN mutation N88K (264COA), a PCR fragment of 118 bp containing exon 2 was amplified. Since the mutation creates a new BseNI site, the wildtype allele remains undigested, whereas the mutant allele yields two fragments (77 and 41 bp). The patient (II/2) as well as the unaffected father (I/1) and brother (II/1) are heterozygous for the mutation N88K (264COA). The mother of the patient does not carry the mutation N88K. (B) Result of a long-range PCR performed in the family with the primers R6 forward (located in intron 5) and rs4282946 reverse (located telomeric to the RAPSN gene). For the father (I/1) and the unaffected brother (II/1) of the patient, the expected wildtype product with a length of 6.7 kb was obtained. For the mother (I/2) and the patient (II/2), only a shorter product of about 2.2 kb was amplified, locating the chromosomal microdeletion between the two primers. Only the patient (II/2) carries both N88K and the microdeletion of the RAPSN gene.
In order to map putative chromosomal deletions/rearrangements, several long-range PCR amplifications were carried out on genomic DNA of the patient using sequences in the second exon of the RAPSN gene (contains mutation 264COA heterozygously) as anchor (Fig. 2). In a PCR with primers RP forward (located centromeric to the RAPSN promoter region) and R2 reverse (located in intron 2 of the RAPSN gene) both alleles were amplified, indicated by heterozygousity for the mutation 264COA. In contrast, a PCR with the primer pair R2 forward (located in intron 1 centromeric to exon 2) and RAPSN 3 0 -UTR reverse (telomeric to the 3 0 -UTR of the RAPSN gene) amplified only the paternal RAPSN allele, as the sequence at position 264 appeared homozygous for
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Fig. 2. (A) Schematic representation of the genomic organization of the RAPSN gene and the position of the deletion characterized in this study. The eight exons of the RAPSN gene are represented by filled boxes and are numbered. Striped boxes symbolize the promoter region and 3 0 -UTR of the gene, repetitive DNA elements are shown as white boxes (only relevant repeat elements are shown). A 4.5 kb portion indicated by gray shading is deleted in the patient. The deletion occurred between the AluSx repeat in intron 6 (centromeric breakpoint) and the L1CM4 repeat in the intergenic region between RAPSN and the single nucleotide polymorphism rs4282946. (B) The upper sequence shows the wildtype sequence (minus strand), the lower the patient’s sequence. The deleted region is indicated by gray shading. The distal AluSq element located upstream the L1CM4 repeat shares 80% homology with the AluSx sequence in intron 6.
A. Therefore, we hypothesized this result may be caused by a deletion or chromosomal rearrangement telomeric to the second RAPSN exon. A long-range PCR with the primers R2 forward (intron 1) and rs4282946 reverse located 3 kb telomeric to RAPSN close to the single nucleotide polymorphism rs4282946 was carried out and revealed the presence of a band of 9 kb, slightly shorter than expected. In the sequencing reaction, this fragment proved to be wildtype at position 264C. This suggested a relatively short chromosomal deletion between exon 2 and the single nucleotide polymorphism rs4282946 rather than a large rearrangement. To delineate the precise deletion breakpoints, subsequent nested PCRs with several primer pairs covering this region converged the area of the deletion. A PCR was carried out in the patient and his family using the forward primer R6 forward (located in intron 5) and the reverse primer rs4282946 reverse. The expected size of the wildtype product is 6.7 kb. However, PCR amplification and agarose gel detection revealed a shorter fragment of 2.2 kb size for both the mother and the patient. This may be due to preferential amplification of the shorter fragment (Fig. 1B). By PCR with the primer pair AluSx forward and L1MC4 reverse (wildtype distance 5.1 kb) a short fragment of 650 bp length was obtained. By sequence comparison the deletion breakpoints were mapped. The telomeric breakpoint is located in the intergenic region between RAPSN and the PSMC3 gene. The centromeric breakpoint
lies within the RAPSN intron 6. The deletion is flanked by a telomeric L1MC4 element (position on chromosome 11: bp 47421072–47421109) and a centromeric AluSx repeat (position on chromosome 11: bp 47425573–47425849). Additional Alu elements (e.g. AluSx and AluSq) are located within the deleted sequence, oriented in different directions. The precise breakpoint is located within a poly(A)stretch shared by the AluSq element adjoining the telomeric breakpoint and the AluSx repeat adjoining the telomeric breakpoint. The total deletion of roughly 4464 bp results in the loss of exons 7 and 8 of RAPSN and premature termination of the transcription product.
4. Discussion To our knowledge, this is the first chromosomal deletion event observed in the RAPSN gene and expands our understanding of the molecular pathogenesis of autosomal-recessive CMS with endplate-deficiency. Up to date, in most patients with CMS due to mutations in the RAPSN gene, the mutation N88K (264COA) has been identified either homozygously, or associated with another heteroallelic mutation. In the patient with sporadic CMS reported in this study, one heterozygous mutation (N88K) of paternal origin, but no second mutation was detected, initially. Extensive molecular analysis of
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the RAPSN gene led to the identification of a compound heterozygous chromosomal deletion of about 4.5 kb disrupting the RAPSN gene. The deletion was inherited from the healthy mother. Numerous human genetic disorders are known to be associated with large deletions of variable size on different chromosomes [22–26]. These deletions may disrupt transcribed genes or may interfere with the expression of genes located distal or proximal to the deletion. For CMS, diseasecausing mutations have been identified in different genes. Most of these mutations are missense, non-sense, splice-site mutations or deletions/insertions of one or a few base pairs, only. However, one chromosomal microdeletion disrupting the 3-AChR gene on chromosome 17p13 (CHRNE D1290) has been described by us, recently [27]. The newly reported mutant allele in a CMS patient carries an extensive deletion of about 4.5 kb on chromosome 11p11.2. Telomeric, no other known genes are affected by the deletion according to public databases. Centromeric, the deletion results in loss of the last two exons (exons 7 and 8) of the RAPSN gene. The consequences of the deletion on mRNA level in the patient’s muscle have not been analyzed as no muscle sample was available. Therefore, the exact consequences of the deletion on open reading frames remain to be determined. Multiple splice products after exon 6 can be envisaged. The mutation might lead to a non-functional protein by affecting the COOH-terminus including the cysteine rich RING-H2 motif (codons 363–402) thought to link rapsyn to the cytoskeletal protein b-dystroglycan [28], and by affecting the C-terminal phosphorylation site sequence (codon 403–406) [29]. Both domains are conserved across species. A dinucleotide CT duplication in exon 7 (1083O1084dupCT) resulting in a frame-shift combined with premature termination at codon 371 has been identified in a CMS patient, recently [21]. However, functional consequences of this mutation have not been reported. In a yeast two-hybrid assay it has been shown that mutant RAPSN constructs lacking the RING-H2 domain (rapsyn1-360-Sos) did not interact with the cytoplasmic domain of b-dystroglycan [28]. When cotransfected with b-dystroglycan in tissue culture, there was no evidence of rapsyn expression nor clustering of b-dystroglycan based on immunofluorescence [28], suggesting that COOHterminally truncated protein is unstable or mislocalized. Another consequence of the microdeletion might be the complete absence of a translated product from the maternal allele due to non-sense-mediated decay of deleted mRNA. In this case, only mRNA carrying the N88K mutation from the paternal allele would be translated into protein. Clinical data reported on CMS patients reveal that severity of disease varies remarkably among patients with RAPSN mutation. Clinical phenotypes range from single episodes of general weakness during childhood infections as the only symptom, to permanent general weakness with frequent exacerbations accompanied by severe respiratory failure. Moreover, two distinct phenotypes, with early and late onset, have been suggested recently [15,30]. Patients with a late onset
phenotype may be asymptomatic in childhood and present as adults with defects of neuromuscular transmission that may be mistaken for seronegative myasthenia [15,18]. However, most patients with RAPSN mutations reported to date exhibit an early onset phenotype [15,17–19,21,30]. Frequently and similar to our patient, first symptoms are evident at birth, arthrogryposis multiplex congenita, recurrent apneas and episodic crisis occur. Early initiation of treatment with anticholinesterase drugs may have successfully prevented further crisis with respiratory insufficiency in our patient. It has been suggested, that the presence of two N88K alleles in a patient may favor a milder, late onset phenotype [21]. In contrast, patients heterozygous for RAPSN N88K harboring a second heteroallelic mutation may be affected more severely. However, homozygous as well as heterozygous N88K patients have been reported with severe phenotypes. Therefore, a clear genotype–phenotype correlation cannot be established, so far. To our knowledge, except for the RAPSN gene no other genes have been mapped into the chromosomal region affected by the reported deletion. This corresponds to the fact that additional phenotypic changes were not found in our patient. Interestingly, the 11p11.2 microdeletion appears to arise from unequal homologous recombination between two Alu repeats (Fig. 2). The centromeric breakpoint was located directly telomeric to an AluSx element that is present in intron 6 of the RAPSN gene. The telomeric breakpoint was found to be adjoined to an AluSq element. Both Alu elements are oriented in sense direction relative to the RAPSN gene, facilitating the unequal homologous recombination. Therefore, we hypothesize that an Alu-mediated deletion underlies the disease-causing rearrangement. Repetitive elements, such as the Alu family of short interspersed nuclear elements contributed to the evolution of the human genome but also to a significant number of human genetic disorders. Two different mechanisms of mutagenesis by Alu elements are known: insertion of Alu elements or unequal homologous recombination. The large number of Alu elements in the human genome provides abundant opportunities for unequal homologous recombination events. Indeed, breakpoint junctions have been frequently found within Alu repeats. Alu-mediated recombinations are responsible for several human diseases including germ-line mutations and somatic mutations in cancer [31]. It is estimated that homologous recombinations between Alu repeats are responsible for up to 0.3% of human genetic diseases [31]. The RAPSN gene harbors two large introns (intron 2 and intron 6) with a length of about 5 and 2.2 kb, respectively. Both introns contain numerous repetitive elements. This situation may favor an increased probability for Alu-mediated deletion or rearrangement events. While methods for the detection of point mutations and small insertions or deletions in genomic DNA are well established, the detection of larger genomic duplications or deletions is more difficult. Mutation scanning methods using PCR as a first step, fail to quantitatively analyze the gene dosage of individual exons. Heterozygous deletions and
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duplications may be overlooked and therefore be underascertained in genetic disorders including CMS. This may be overcome by the development of new techniques allowing for a quantitation [32]. Numerous interspersed Alu elements may predispose the RAPSN locus for genetic rearrangements. Therefore, we emphasize that comprehensive mutation analysis of the RAPSN gene should also include scanning methods for large insertions, deletions, and duplications.
Acknowledgements We thank the patient and his family for participating in this study. We thank Ursula Klutzny and Petra Mitzscherling for expert assistance. This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) and the Deutsche Gesellschaft fu¨r Muskelkranke (DGM) to HL and AA, and by a grant from the Saxonian State Ministry to AH. JSM receives a scholarship from the Boehringer Ingelheim Fonds.
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Case report
A newly identified chromosomal microdeletion of the rapsyn gene causes a congenital myasthenic syndrome Juliane S. Mu¨llera,1, Angela Abichta,1, Hans-Ju¨rgen Christenb, Rolf Stuckaa, Ulrike Scharac, Wilhelm Mortierd, Angela Huebnere, Hanns Lochmu¨llera,* a
Department of Neurology and Gene Center, Friedrich-Baur-Institute, Ludwig-Maximilians-University, Munich, Germany b Department of Neuropediatrics, Kinderkrankenhaus auf der Bult, Hannover, Germany c Department of Neuropediatrics, Staedtische Kliniken, Neuss, Germany d Department of Pediatrics and Pediatric Neurology, Ruhr-University of Bochum, Bochum, Germany e Department of Pediatrics, Technical University Dresden, Dresden, Germany Received 29 January 2004; received in revised form 1 June 2004; accepted 9 June 2004
Abstract The objective is mutation analysis of the RAPSN gene in a patient with sporadic congenital myasthenic syndrome (CMS). Mutations in various genes encoding proteins expressed at the neuromuscular junction may cause CMS. Most mutations affect the epsilon subunit gene of the acetylcholine receptor (AChR) leading to endplate AChR deficiency. Recently, mutations in the RAPSN gene have been identified in several CMS patients with AChR deficiency. In most patients, RAPSN N88K was identified, either homozygously or heteroallelic to a second missense mutation. A sporadic CMS patient from Germany was analyzed for RAPSN mutations by RFLP, long-range PCR and sequence analysis. Clinically, the patient presents with an early onset CMS, associated with arthrogryposis multiplex congenita, recurrent episodes of respiratory insufficiency provoked by infections, and a moderate general weakness, responsive to anticholinesterase treatment. The mutation RAPSN N88K was found heterozygously to a large deletion of about 4.5 kb disrupting the RAPSN gene. Interestingly, an Alu-mediated unequal homologous recombination may have caused the deletion. We hypothesize that numerous interspersed Alu elements may predispose the RAPSN locus for genetic rearrangements. q 2004 Elsevier B.V. All rights reserved. Keywords: Congenital myasthenic syndrome; Rapsyn; Mutation N88K; Chromosomal deletion; Neuromuscular junction
1. Introduction Congenital myasthenic syndromes (CMS) are a heterogeneous group of disorders, where neuromuscular transmission is impaired by different inherited defects [1]. Molecular genetic studies led to the identification of numerous pathogenic mutations in various genes, encoding presynaptic (choline acetyltransferase, ChAT) [2–5], synaptic (collagenic tail subunit of acetylcholinesterase, ColQ) [6–11], and postsynaptic proteins (subunits of acetylcholine
* Corresponding author. Address: Genzentrum Mu¨nchen, Feodor-LynenStr. 25, 81377 Mu¨nchen, Germany. Tel.: C49-89-2180-76887; fax: C4989-2180-76999. E-mail address: [email protected] (H. Lochmu¨ller). 1 These authors contributed equally to this work. 0960-8966/$ - see front matter q 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2004.06.010
receptor, AChR and rapsyn) [12]. The majority of CMS affect postsynaptic functions. Mutations in AChR subunit genes alter AChR channel kinetics and/or cause endplate (EP-) AChR deficiency [12]. Especially mutations in the gene CHRNE encoding the 3 subunit of the AChR are responsible for CMS with EP-AChR deficiency [13,14]. In addition, mutations in the RAPSN gene have been shown to cause postsynaptic CMS with EP-AChR deficiency [15–21]. Rapsyn (acetylcholine receptor-associated protein of the synapse), a peripheral membrane protein of skeletal muscle is essential for clustering nicotinic acetylcholine receptors at high density in the postsynaptic membrane. In the great majority of CMS patients with underlying RAPSN mutations, a specific missense mutation of the gene (RAPSN N88K) has been identified homozygously or compound heterozygously to a second point mutation
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(missense, frame-shift, stop, splice-site or promoter mutations). Here we report the first chromosomal microdeletion of the RAPSN gene.
2. Material and methods Patients, DNA samples. Venous blood samples were obtained from the CMS patient as well as from his unaffected brother and unaffected parents. All studies were carried out with informed consent of the patient’s parents and were approved by the institutional ethics review board. Genomic DNA was isolated using a blood and tissue culture DNA extraction kit according to the manufacturer’s recommendations (Wizard Genomic DNA Purification Kit, Promega, Mannheim, Germany). Sequence analysis, RFLPs. PCR primers were designed based on the published genomic structure [GenBank accession number AC090559/gi:22002211, mRNA AF449218/gi19310212] of the RAPSN gene. Nucleotide positions are given according to the genome assembly July 2003 of the University of California Santa Cruz Genome Bioinformatics (http://genome.ucsc.edu). The Repeat Masker program provided by this website was used to analyze the nature of the sequences flanking the deletion breakpoints. In the patient, all eight exons, flanking intronic regions, and the promoter of the RAPSN gene were amplified by PCR and sequenced. PCR-amplified fragments were purified with the NucleoSpin Extract kit (Macherey– Nagel, Du¨ren, Germany) and sequenced with an Applied Biosystems model 3100 Avant DNA sequencer and fluorescein-labeled dideoxy terminators (Perkin–Elmer, Foster City, CA, USA). Screening for the mutation N88K (264COA) in exon 2 of the RAPSN gene was performed as described previously [18]. A 118 bp fragment in exon 2 was amplified by PCR using primers 5 0 -gaggatgccgacttcctcctgg3 0 and 5 0 -tggtaccaggcagcccaaggc-3 0 . The mutation N88K (264COA) creates a new BseNI site. In addition, all 12 exons, adjacent intronic regions and the promotor region of the AChR 3-subunit gene [CHRNE, GenBank accession number AF105999/gi4580858] were amplified by PCR and sequenced to exclude a CHRNE mutation in the patient. Genotype and polymorphism analysis. For haplotype analysis five polymorphic microsatellite markers on
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chromosome 11p11.2 flanking the RAPSN gene (D11S986, D11S1344, D11S4109, D11S4076, D11S1883) were chosen based on information obtained from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) and the University of California Santa Cruz Genome Bioinformatics (http://genome.ucsc.edu). Markers were amplified by PCR using fluorescence-labeled 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, USA). Determination of the deletion breakpoints. The longrange polymerase chain reaction was carried out on genomic DNA of the patient and his family using the Expand Long Template PCR System from Roche (Basel, Switzerland). A panel of specific primers in sense and antisense direction was used to determine the deletion breakpoints (Table 1).
3. Results Clinical data. The German patient, currently 3 years of age, is the second child of non-consanguineous healthy parents. His 17-months-old brother is healthy. The index patient was born after a 36-week pregnancy. The mother noted decreased fetal movements. The spontaneous vaginal delivery was complicated by a breech presentation. Postpartum, multiple hematomas and an arthrogryposis multiplex congenita, with multiple joint contractures of upper and lower limbs were reported. At day 3 postpartum, assisted ventilation was required for a few days. Since birth, severe difficulties in feeding and sucking were observed that prevented breast-feeding. Moreover, an open mouth and intermittent strabism were noted. Repeatedly, viral infections of the upper airways led to abnormal fatigue, weakness and respiratory insufficiency that required hospitalization. At the age of 15 months, a daytime-dependent eyelid ptosis was observed. Whereas cognitive development was normal, motor milestones were moderately delayed. Overall, only a slow and gradual improvement of the general condition occurred until a neuromuscular transmission defect was diagnosed and specific therapy was initiated. The patient was diagnosed with CMS at the age of 20 months, when a respiratory infection was accompanied
Table 1 Oligonucleotide sequences Name
Sequence
Position
RP forward R2 forward R2 reverse R6 forward AluSx forward 3 0 -UTR reverse L1CM4 reverse rs4282946 reverse
5 0 -TTGCCCTGGGGCAGGAAGAAGC 5 0 -AGGCTGGGGTCCAAGGCTCAGAGT 5 0 -GCCACAGGGTGTGTGCCTCA 5 0 -ACCCTGTGCTTCCCTGTGAGCA 5 0 -ACCAACATGGTGAAACTCT 5 0 -CCACCCACTGCACGTCAGCTTC 5 0 -TGAAATCCATGAGTTCACA 5 0 -GAGCCCATCCAAGCTGGCTCC
RAPSN promoter region RAPSN intron 1 RAPSN intron 2 RAPSN intron 5 RAPSN intron 6 Telomeric to RAPSN 3 0 -UTR Between rs4282946 and L1CM4 telomeric to SNP rs4282946
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by sudden development of severe respiratory insufficiency requiring assisted ventilation for 1 week. At the age of 21 months, the general physical and neurological examination revealed mild bilateral ptosis with mild restriction of extraocular movements in all directions of gaze. Bulbar weakness was apparent by a weak cough and involvement of facial muscles. In addition, the face appeared slightly elongated. The patient’s mouth was opened and the palate appeared to be high arched. Moderate contractures of knee and elbow joints were noted. Exercise-induced, generalized weakness of limb muscles, predominantly involving proximal muscles, was also noted. The child was able to stand with assistance, but was not yet able to walk unassisted. The anti-AChR antibody test was negative. Repetitive stimulation (3/s) of two distal (right N. medianus and right N. ulnaris) as well as two proximal motor nerves (right and left N. accessorius) revealed no decremental response of the compound muscle action potential at rest. An intravenous edrophonium (CamsilonR) test was unequivocally positive. A therapy with pyridostigmine bromide (MestinonR) at a moderate dosage (2.4 mg/kg per day) was started and resulted in immediate and significant clinical improvement of ptosis, motor skills and behavior. In particular, the child was able to walk independently only one day after treatment was initiated. His subsequent clinical course has been stable, with further improvements in motor development. Recurrent infections were successfully managed by intermittent increase of the pyridostigmine dosage or by decrease of the dosing intervals, respectively. Moreover, a slow, but steady regression of the contractures was seen. Mutational analysis. Sequence analysis of the coding and the promoter region of the RAPSN gene revealed a heterozygous C-to-A transition in exon 2 at nucleotide position 264 (264COA) in the index patient II/2 of our German CMS pedigree. The mutation leads to an amino acid exchange (N88K) that has previously been described by us and others as a recessive CMS mutation. Restriction analysis (Fig. 1A) revealed that unaffected family members are either carrying the mutation N88K heterozygously (I/1 and II/1) or do not carry the mutation (I/2). This is in accordance with an autosomal-recessive trait. However, no additional mutation was found by PCR-based sequence analysis of the coding and promoter region of the patient’s RAPSN gene. Therefore, we hypothesized that a chromosomal deletion or a chromosomal rearrangement of the maternal RAPSN allele may have been missed by our analysis. Identification and characterization of the deletion. Haplotype analysis using five polymorphic microsatellite markers on chromosome 11p11.2 flanking the RAPSN gene (D11S986, D11S1344, D11S4109, D11S4076, D11S1883) excluded an extensive chromosomal rearrangement. In addition, maternity of the patient’s mother was verified since the patient and his mother share the same allele (data not shown). Haplotype analysis confirmed that the index patient and his unaffected brother both inherited the N88K allele from their father.
Fig. 1. (A) Restriction enzyme analysis in the CMS family. To detect the RAPSN mutation N88K (264COA), a PCR fragment of 118 bp containing exon 2 was amplified. Since the mutation creates a new BseNI site, the wildtype allele remains undigested, whereas the mutant allele yields two fragments (77 and 41 bp). The patient (II/2) as well as the unaffected father (I/1) and brother (II/1) are heterozygous for the mutation N88K (264COA). The mother of the patient does not carry the mutation N88K. (B) Result of a long-range PCR performed in the family with the primers R6 forward (located in intron 5) and rs4282946 reverse (located telomeric to the RAPSN gene). For the father (I/1) and the unaffected brother (II/1) of the patient, the expected wildtype product with a length of 6.7 kb was obtained. For the mother (I/2) and the patient (II/2), only a shorter product of about 2.2 kb was amplified, locating the chromosomal microdeletion between the two primers. Only the patient (II/2) carries both N88K and the microdeletion of the RAPSN gene.
In order to map putative chromosomal deletions/rearrangements, several long-range PCR amplifications were carried out on genomic DNA of the patient using sequences in the second exon of the RAPSN gene (contains mutation 264COA heterozygously) as anchor (Fig. 2). In a PCR with primers RP forward (located centromeric to the RAPSN promoter region) and R2 reverse (located in intron 2 of the RAPSN gene) both alleles were amplified, indicated by heterozygousity for the mutation 264COA. In contrast, a PCR with the primer pair R2 forward (located in intron 1 centromeric to exon 2) and RAPSN 3 0 -UTR reverse (telomeric to the 3 0 -UTR of the RAPSN gene) amplified only the paternal RAPSN allele, as the sequence at position 264 appeared homozygous for
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Fig. 2. (A) Schematic representation of the genomic organization of the RAPSN gene and the position of the deletion characterized in this study. The eight exons of the RAPSN gene are represented by filled boxes and are numbered. Striped boxes symbolize the promoter region and 3 0 -UTR of the gene, repetitive DNA elements are shown as white boxes (only relevant repeat elements are shown). A 4.5 kb portion indicated by gray shading is deleted in the patient. The deletion occurred between the AluSx repeat in intron 6 (centromeric breakpoint) and the L1CM4 repeat in the intergenic region between RAPSN and the single nucleotide polymorphism rs4282946. (B) The upper sequence shows the wildtype sequence (minus strand), the lower the patient’s sequence. The deleted region is indicated by gray shading. The distal AluSq element located upstream the L1CM4 repeat shares 80% homology with the AluSx sequence in intron 6.
A. Therefore, we hypothesized this result may be caused by a deletion or chromosomal rearrangement telomeric to the second RAPSN exon. A long-range PCR with the primers R2 forward (intron 1) and rs4282946 reverse located 3 kb telomeric to RAPSN close to the single nucleotide polymorphism rs4282946 was carried out and revealed the presence of a band of 9 kb, slightly shorter than expected. In the sequencing reaction, this fragment proved to be wildtype at position 264C. This suggested a relatively short chromosomal deletion between exon 2 and the single nucleotide polymorphism rs4282946 rather than a large rearrangement. To delineate the precise deletion breakpoints, subsequent nested PCRs with several primer pairs covering this region converged the area of the deletion. A PCR was carried out in the patient and his family using the forward primer R6 forward (located in intron 5) and the reverse primer rs4282946 reverse. The expected size of the wildtype product is 6.7 kb. However, PCR amplification and agarose gel detection revealed a shorter fragment of 2.2 kb size for both the mother and the patient. This may be due to preferential amplification of the shorter fragment (Fig. 1B). By PCR with the primer pair AluSx forward and L1MC4 reverse (wildtype distance 5.1 kb) a short fragment of 650 bp length was obtained. By sequence comparison the deletion breakpoints were mapped. The telomeric breakpoint is located in the intergenic region between RAPSN and the PSMC3 gene. The centromeric breakpoint
lies within the RAPSN intron 6. The deletion is flanked by a telomeric L1MC4 element (position on chromosome 11: bp 47421072–47421109) and a centromeric AluSx repeat (position on chromosome 11: bp 47425573–47425849). Additional Alu elements (e.g. AluSx and AluSq) are located within the deleted sequence, oriented in different directions. The precise breakpoint is located within a poly(A)stretch shared by the AluSq element adjoining the telomeric breakpoint and the AluSx repeat adjoining the telomeric breakpoint. The total deletion of roughly 4464 bp results in the loss of exons 7 and 8 of RAPSN and premature termination of the transcription product.
4. Discussion To our knowledge, this is the first chromosomal deletion event observed in the RAPSN gene and expands our understanding of the molecular pathogenesis of autosomal-recessive CMS with endplate-deficiency. Up to date, in most patients with CMS due to mutations in the RAPSN gene, the mutation N88K (264COA) has been identified either homozygously, or associated with another heteroallelic mutation. In the patient with sporadic CMS reported in this study, one heterozygous mutation (N88K) of paternal origin, but no second mutation was detected, initially. Extensive molecular analysis of
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the RAPSN gene led to the identification of a compound heterozygous chromosomal deletion of about 4.5 kb disrupting the RAPSN gene. The deletion was inherited from the healthy mother. Numerous human genetic disorders are known to be associated with large deletions of variable size on different chromosomes [22–26]. These deletions may disrupt transcribed genes or may interfere with the expression of genes located distal or proximal to the deletion. For CMS, diseasecausing mutations have been identified in different genes. Most of these mutations are missense, non-sense, splice-site mutations or deletions/insertions of one or a few base pairs, only. However, one chromosomal microdeletion disrupting the 3-AChR gene on chromosome 17p13 (CHRNE D1290) has been described by us, recently [27]. The newly reported mutant allele in a CMS patient carries an extensive deletion of about 4.5 kb on chromosome 11p11.2. Telomeric, no other known genes are affected by the deletion according to public databases. Centromeric, the deletion results in loss of the last two exons (exons 7 and 8) of the RAPSN gene. The consequences of the deletion on mRNA level in the patient’s muscle have not been analyzed as no muscle sample was available. Therefore, the exact consequences of the deletion on open reading frames remain to be determined. Multiple splice products after exon 6 can be envisaged. The mutation might lead to a non-functional protein by affecting the COOH-terminus including the cysteine rich RING-H2 motif (codons 363–402) thought to link rapsyn to the cytoskeletal protein b-dystroglycan [28], and by affecting the C-terminal phosphorylation site sequence (codon 403–406) [29]. Both domains are conserved across species. A dinucleotide CT duplication in exon 7 (1083O1084dupCT) resulting in a frame-shift combined with premature termination at codon 371 has been identified in a CMS patient, recently [21]. However, functional consequences of this mutation have not been reported. In a yeast two-hybrid assay it has been shown that mutant RAPSN constructs lacking the RING-H2 domain (rapsyn1-360-Sos) did not interact with the cytoplasmic domain of b-dystroglycan [28]. When cotransfected with b-dystroglycan in tissue culture, there was no evidence of rapsyn expression nor clustering of b-dystroglycan based on immunofluorescence [28], suggesting that COOHterminally truncated protein is unstable or mislocalized. Another consequence of the microdeletion might be the complete absence of a translated product from the maternal allele due to non-sense-mediated decay of deleted mRNA. In this case, only mRNA carrying the N88K mutation from the paternal allele would be translated into protein. Clinical data reported on CMS patients reveal that severity of disease varies remarkably among patients with RAPSN mutation. Clinical phenotypes range from single episodes of general weakness during childhood infections as the only symptom, to permanent general weakness with frequent exacerbations accompanied by severe respiratory failure. Moreover, two distinct phenotypes, with early and late onset, have been suggested recently [15,30]. Patients with a late onset
phenotype may be asymptomatic in childhood and present as adults with defects of neuromuscular transmission that may be mistaken for seronegative myasthenia [15,18]. However, most patients with RAPSN mutations reported to date exhibit an early onset phenotype [15,17–19,21,30]. Frequently and similar to our patient, first symptoms are evident at birth, arthrogryposis multiplex congenita, recurrent apneas and episodic crisis occur. Early initiation of treatment with anticholinesterase drugs may have successfully prevented further crisis with respiratory insufficiency in our patient. It has been suggested, that the presence of two N88K alleles in a patient may favor a milder, late onset phenotype [21]. In contrast, patients heterozygous for RAPSN N88K harboring a second heteroallelic mutation may be affected more severely. However, homozygous as well as heterozygous N88K patients have been reported with severe phenotypes. Therefore, a clear genotype–phenotype correlation cannot be established, so far. To our knowledge, except for the RAPSN gene no other genes have been mapped into the chromosomal region affected by the reported deletion. This corresponds to the fact that additional phenotypic changes were not found in our patient. Interestingly, the 11p11.2 microdeletion appears to arise from unequal homologous recombination between two Alu repeats (Fig. 2). The centromeric breakpoint was located directly telomeric to an AluSx element that is present in intron 6 of the RAPSN gene. The telomeric breakpoint was found to be adjoined to an AluSq element. Both Alu elements are oriented in sense direction relative to the RAPSN gene, facilitating the unequal homologous recombination. Therefore, we hypothesize that an Alu-mediated deletion underlies the disease-causing rearrangement. Repetitive elements, such as the Alu family of short interspersed nuclear elements contributed to the evolution of the human genome but also to a significant number of human genetic disorders. Two different mechanisms of mutagenesis by Alu elements are known: insertion of Alu elements or unequal homologous recombination. The large number of Alu elements in the human genome provides abundant opportunities for unequal homologous recombination events. Indeed, breakpoint junctions have been frequently found within Alu repeats. Alu-mediated recombinations are responsible for several human diseases including germ-line mutations and somatic mutations in cancer [31]. It is estimated that homologous recombinations between Alu repeats are responsible for up to 0.3% of human genetic diseases [31]. The RAPSN gene harbors two large introns (intron 2 and intron 6) with a length of about 5 and 2.2 kb, respectively. Both introns contain numerous repetitive elements. This situation may favor an increased probability for Alu-mediated deletion or rearrangement events. While methods for the detection of point mutations and small insertions or deletions in genomic DNA are well established, the detection of larger genomic duplications or deletions is more difficult. Mutation scanning methods using PCR as a first step, fail to quantitatively analyze the gene dosage of individual exons. Heterozygous deletions and
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duplications may be overlooked and therefore be underascertained in genetic disorders including CMS. This may be overcome by the development of new techniques allowing for a quantitation [32]. Numerous interspersed Alu elements may predispose the RAPSN locus for genetic rearrangements. Therefore, we emphasize that comprehensive mutation analysis of the RAPSN gene should also include scanning methods for large insertions, deletions, and duplications.
Acknowledgements We thank the patient and his family for participating in this study. We thank Ursula Klutzny and Petra Mitzscherling for expert assistance. This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) and the Deutsche Gesellschaft fu¨r Muskelkranke (DGM) to HL and AA, and by a grant from the Saxonian State Ministry to AH. JSM receives a scholarship from the Boehringer Ingelheim Fonds.
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