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- congenital myasthenic syndrome
- epsilon subunit of the acetylcholine receptor
- neuromuscular junction
Acetylcholine receptor (AChR) deficiency is the most common form of congenital myasthenic syndrome (CMS). Most AChR deficiencies are caused by mutations in the coding region of the AChR epsilon subunit.1 We report an Iranian Muslim family from the province of Eastern Azerbaijan (Maragheh) in which three of five offspring of consanguineous parents had early onset CMS arising from a newly described mutation in the epsilon subunit of the AChR; this mutation has been identified homozygously in all the three sibs. This is the first report of an AChR epsilon subunit mutation in Iran.
The affected brother (case 1) was 23 years old and the affected sisters were 19 (case 2) and 16 (case 3) years old. The unaffected brother was 21 and the healthy sister was 13 years old. There was no history of miscarriage or infant mortality in the family, although, according to verbal accounts from the mother, fetal movements were decreased in all the affected sibs and case 1 had a difficult labour. The clinical diagnosis of CMS in this family had been made first in 1996. All cases presented with neonatal hypotonia, regurgitation, ptosis (case 1 developed ptosis at the age of 6 months), and delayed motor milestones. The course of the disease had been slowly progressive, transiently exacerbated by stress. Generalised weakness was more severe in the afternoons. Treatment with prednisone for 1 year yielded no improvement. There was positive response to pyridostigmine bromide (Mestinon) and the required doses had gradually been increased. There were no other affected relatives in the family.
On clinical examination, all patients were ambulant, although with limited walking distance. There was bilateral ptosis and limited eye movements. The bulbar muscles were also involved, manifesting as nasal speech (more severe in case 1), chewing problems (in cases 1 and 3), swallowing difficulties, and bilateral facial weakness. Pupillary response to light was normal. Muscle involvement was predominantly proximal. Cases 1 and 3 had a waddling gait, especially in the afternoons. Deep tendon reflexes were normal. There was no muscle wasting, scapulae alatae, scoliosis, or respiratory distress. No hospitalisations for respiratory problems were reported. Case 1 had left elbow hyperlaxity and case 3 also revealed severe bilateral elbow hyperlaxity (more severe on the left side). Case 3 had mild flexion contracture of her left knee.
Muscle enzymes were normal. Electromyography of nasalis muscles showed decrements (60%, 53%, and 35% in cases 1, 2, and 3, respectively) in response to low frequency repetitive nerve stimulation. Anti-AChR antibodies were negative in all affected sibs.
All 12 exons, adjacent intronic regions, and the promoter region of the AChR epsilon subunit gene (CHRNE, GenBank accession number AF105999/gi4580858) were amplified by PCR. PCR amplified fragments were purified with the NucleoSpin Extract kit (Macherey-Nagel, Düren, Germany) and sequenced with an Applied Biosystems model 3100 Avant DNA sequencer and fluorescence labelled dideoxy terminators (Perkin-Elmer, Foster City, CA, USA).
Screening for mutation K171X of the CHRNE gene was performed by restriction digest of PCR products in the patients and the healthy siblings. A 304 bp fragment containing exon 6 was amplified by PCR from genomic DNA using primers 5′-AGGTACAGATGGGAACAGAG-3′ and 5′-TCTGGACCCCGTCTAGAAGCG-3′. A BfaI digest yields 218, 71, and 15 bp fragments for the wild type allele. The mutation K171X introduces a new BfaI restriction site, therefore resulting in fragments of 153, 71, 65, and 15 bp in length (fig 1).
Analysis of the CHRNE gene revealed a homozygous nonsense mutation K171X (511A→T) in all three affected siblings. The two healthy siblings did not carry this mutation. DNA of the parents was not available for genetic analysis. The mutation causes a premature translation stop in exon 6 of the epsilon subunit of the AChR. Position 171 is located in the N-terminal extracellular domain of the epsilon subunit protein. The mutation has not been previously described in the literature.
Severe endplate AChR deficiency can result from different types of recessive mutations in the AChR subunit genes. As the mutation identified in this family lies in the N-terminal region of the epsilon subunit, a putative translation product would not be inserted into the membrane and expressed at the cell surface. Another possibility is the degradation of the mutated epsilon subunit mRNA containing a premature stop codon in exon 6 by nonsense mediated decay. Therefore, we hypothesise that the K171X mutation leads to a deficiency of AChR at the endplate.
Mutations in the coding region of the AChR epsilon subunit are said to be a common cause of CMS in eastern Mediterranean countries.2 Most of the CHRNE mutations reported so far are null mutations leading to receptor deficiency at the endplate. However, cases of homozygous nonsense mutations, as reported in our case, are rather rare compared to the number of frameshift and missense mutations. AChR epsilon subunit mutations have not yet been reported from Iran, despite the high rate of consanguineous marriages in the country. CMS associated with facial malformations has been reported in Iranian and Iraqi Jews,3 who were subsequently found to have a homozygous mutation (–38A→G) in an E-box element within the promoter region of the RAPSN gene.4 Another similar patient of Iranian Jewish origin has been reported with the same founder mutation in a series of early onset CMS cases.1
The clinical features of our cases with CHRNE K171X mutation are similar to those of other patients, including Europeans, with AChR epsilon mutations.1 Although congenital joint contractures have not been previously reported in CMS patients with AChR epsilon mutations, one of our cases (case 3) had mild flexion contracture of her left knee, although, in contrast to patients with RAPSN mutations, none of our cases had arthrogryposis multiplex congenita.1,5 A distinguishing feature in cases 1 and 3 was the presence of asymmetric elbow hyperlaxity. Joint laxity has not been previously reported in any type of CMS; however, it can be seen in some congenital myopathies. We postulate that fetal hypotonia in the presence of intrauterine biomechanical forces might have influenced the normal modelling of the elbow joint.
It seems that the incidence of CMS in Iran is similar to that in other countries. Recognition of differing features of CMS could help establish a definite genetic diagnosis and help implement appropriate measures.
We thank the patients and their family for their kind participation in this study.
This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) to HL and AA. JSM receives a scholarship from the Boehringer Ingelheim Fonds.
Competing interests: none declared
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