Independent CHRNE mutations at serine 503 in English Springer Spaniels and a Smooth Fox Terrier having congenital myasthenic syndrome

Congenital myasthenic syndromes (CMSs) are inherited disorders of neuromuscular transmission. In the 1980s, spontaneously occurring CMS following autosomal recessive inheritance patterns were described in English Springer Spaniels (ESSs) (Oda et al., 1984) and Smooth Fox Terriers (SFTs) (Miller et al., 1983; OMIA:000685–9615). Affected puppies exhibited muscle weakness and fatigability that was exacerbated by exercise. Skeletal muscle biopsies revealed notably fewer acetylcholine receptors (AChRs) than healthy controls, and no autoantibodies against AChR were detected. Ohno et al. (2023) describe 35 genes classified into 14 groups according to the pathomechanical, clinical, and therapeutic features of human CMS patients. Forms of CMS with AChR deficiency most often result from mutation of CHRNE, encoding the epsilon subunit of the AChR (Finsterer, 2019). Homozygous mutations in other subunits are typically embryonic lethal (Engel et al., 2015). To identify the genetic cause for CMS in these breeds, we sequenced the coding regions and splice sites of CHRNE.

We obtained archival thymus tissue from two affected ESS half-siblings, peripheral blood leukocytes from their unaffected dam (an obligate carrier), and cultured muscle cells from an affected SFT. Buccal swabs were collected from 17 unaffected, unrelated ESSs with informed owner consent under protocols approved by the Clemson University Institutional Review Board (IBC2015-24). DNA was extracted following Puregene Kit protocols (Qiagen). PCR amplification and sequencing of CHRNE exons 3–13 were conducted for the affected individuals and obligate carrier as described in Rinz et al., 2015. Based on updated gene annotation (XM_014113502.3), we designed new primers to capture exons 1 and 2: exon 1 forward 5′-GAATCATCGGTGGAATCTGT-3′ and reverse 5′-GGAGTAGAAATGAGAGGGACC-3′, exon 2 forward 5′-CAATGATGAGTTTTCTGGGTG-3′ and reverse 5′-CCAATCACACCAGCAGAGTC-3′. Resultant sequences were compared to the canFam4 reference genome.

In both breeds, we discovered unique point mutations at position chr5:31915101 in exon 13. A C>A transversion in the ESS predicts the substitution of an arginine for a serine (S503R) (XP_013968977.2). A rapid genotyping protocol was developed through restriction enzyme digestion using Hinf1 (Fisher BioReagents), following manufacturer's instructions. PCR products from exons 12 and 13 incubated with Hinf1 resulted in distinct banding patterns corresponding to genotype when resolved on an agarose gel (Figure S1). All 17 healthy ESSs produced a single 612-bp band. The two affected ESSs produced bands at 398 and 214 bp, and the obligate carrier had all three fragment sizes.

In the affected SFT, we identified a 1-bp insertion (c.1508_1509insG) that predicts a frameshift mutation, p.Ser503Argfs*14. The ESS and SFT variants were absent from 1987 dog genomes available through Dog10K (Meadows et al., 2023) and 624 additional dog genomes (Bell et al., 2023). The databases contained 9 SFTs and 4 ESSs. In 2017, the SFT variant was described in a Heideterrier having CMS (Herder et al., 2017). Phenotypic similarity between these hunting breeds suggests the pathogenic allele may be shared identical by descent. A lack of modern CMS cases in the SFT and ESS suggests that these alleles were eliminated or remain at very low frequencies. CMS in the dog has now been attributed to six unique variants in three genes (Table 1).

Erin Peterson: Data curation; formal analysis; funding acquisition; visualization; writing – review and editing. Tori E. Rudolph: Data curation; formal analysis; visualization; writing – original draft; writing – review and editing. Alison Starr-Moss: Data curation; writing – review and editing. Kendall Anderson: Data curation; writing – review and editing. Vanda A. Lennon: Conceptualization; data curation; writing – review and editing. G. Diane Shelton: Writing – review and editing. Leigh Anne Clark: Conceptualization; funding acquisition; project administration; supervision; writing – original draft; writing – review and editing.

This work was funded in part by the Clemson University Honors College.

The authors have no conflicts of interest to declare.