Association of the Gene Encoding the Δ-Subunit of the Muscle

BRIEF COMMUNICATION Association of the gene encoding the d-subunit of the muscle acetylcholine receptor (CHRND) with acquired autoimmune myasthenia gravis

M Giraud1, B Eymard2, C Tranchant3, P Gajdos4 and H-J Garchon1 1INSERM U580, Hoˆpital Necker, Paris, France; 2Service de Neurologie, Institut de Myologie, Hoˆpital de la Salpeˆtrie`re, Paris; 3Service de Neurologie, Hoˆpital Civil, CHRU Strasbourg, France; 4Service de Re´animation, Hoˆpital Raymond Poincare´, Garches, France

The muscle acetylcholine receptor (AChR) is the main target self-antigen in acquired autoimmune myasthenia gravis (MG). Here, we investigated an association of MG with the CHRND gene encoding the d-subunit of the AChR. Using a microsatellite repeat located in the second intron of the gene, we observed a preferential transmission of the allele 268 in 114 one-generation families with one myasthenic child (Pc ¼ 0.0154). This allele was also over-represented in a group of 350 unrelated nonthymoma MG patients (OR ¼ 1.78, P ¼ 0.038), but not in 84 thymoma patients, compared to 168 healthy controls. Moreover, among nonthymoma patients, those lacking serum anti-titin antibodies appeared to be best associated (OR ¼ 2.07, P ¼ 0.017). In contrast, there was no distortion in the transmission of a single-nucleotide substitution polymorphisms (SNPs) in the 30 untranslated region of CHRND nor in that of two SNPs located in the closely linked CHRNG gene, 4.5 kb telomeric to CHRND. The data warrant a detailed investigation of CHRND polymorphism in MG patients. Genes and Immunity (2004) 5, 80–83. doi:10.1038/sj.gene.6364041

Keywords: myasthenia gravis; genetics; CHRND; autoimmunity; acetylcholine receptor

Acquired autoimmune myasthenia gravis (MG) is an pathogenesis.14,15 Finally, the importance of the d-subunit autoimmune disorder of the neuromuscular junction in muscle AChR structure and function was demon- resulting in fatigability of striated muscles.1–3 In most strated by the observation of congenital myasthenic patients, it is caused by autoantibodies directed at the syndromes that are caused by mutations in the CHRND muscle acetylcholine receptor (AChR), a pentameric gene.16,17 molecular complex made of four different subunits with The CHRND gene is located on chromosome 2q37.1 stoichiometry a2bde in adult or a2bgd in embryonic and consists of 12 exons spanning 9.3 kb (Figure 1). To muscle.4,5 As is the case for most autoimmune diseases, investigate an association with MG, we selected a (GA)n MG has a complex genetic control.6 An HLA-linked locus microsatellite repeat, CHRND_MS, located in its second has been characterized, but it explains the disease intron (The Genome Database: GDB:439020) and three susceptibility only partly.7 Given the central role of single nucleotide substitution polymorphisms (SNPs) AChR in MG pathogenesis, the genes that encode AChR that were described in dbSNP (build 96). Two of the subunits are candidates for an analysis of the genetic SNPs, rs1004432 and rs1550093, are in the last intron and factors that predispose to MG. On this basis, association the third one, rs2767, is in the 30 untranslated region. and linkage of CHRNA1, the gene for the a-subunit of Genotyping of the CHRND_MS microsatellite was the AChR, were demonstrated previously.8,9 conducted as described,18 except that one of the Here, we have investigated the role of the CHRND oligonucleotide primers that was used for amplification gene that encodes the d-subunit of the AChR. This gene was tagged with a fluorescent label. Amplification is also a candidate of great interest. The d-subunit products were migrated on an ABI 373XL automated displays immunogenic epitopes for both B and T sequencer (Applied Biosystems, Foster City, CA, USA). cells.10–12 It interacts with the a-subunit to form a Alleles were determined with the Genescan and Geno- heterodimer in the first step of AChR assembly in typer software and designated by their size in base pairs. muscle cells.13 In contrast, it is expressed at a very low SNPs were characterized by multiplexing single-base level in the thymus and it seems to be excluded from the chain extension reactions, using the SNaPshot system AChR complexes that are formed in this organ, which (Applied Biosystems). All markers were in Hardy– plays an essential role in tolerance induction and in MG Weinberg equilibrium. A transmission-disequilibrium test (TDT)19 was performed in 114 one-generation Caucasoid families with Correspondence: Dr HJ Garchon, INSERM U580, Hoˆpital Necker, 161 rue one child affected with MG. Transmission was assessed de Se`vres, 75743 Paris Cedex 15, France. E-mail: [email protected] as implemented in the Genehunter software.20 Cases Received 05 March 2003; revised 07 October 2003 with one missing parent were included only when the CHRND and myasthenia gravis M Giraud et al 81

Figure 1 Polymorphisms in the CHRND and CHRNG genes. Markers selected for the association study are shown by vertical bars. Transcriptional orientation of the genes is indicated by arrows. Open rectangles depict exons. Locus information and physical distances were obtained from the Ensembl web server (http://www.ensembl.org). Linkage disequilibrium between pairs of markers (square brackets) was estimated with the D0 measure, shown above the parentheses, using the expectation-maximization algorithm, as implemented in the LDMAX software (http://www.sph.umich.edu/csg/abecasis/GOLD/).

genotyped parent and the proband were both distinct Table 1 Transmission of parental CHRND_MS microsatellite heterozygotes.21 For 64 patients, both parents could be alleles to MG offspring genotyped. For 50 other patients, one parent was available. Altogether, 98 parents were heterozygous for Allele Transmitted, N (%) Untransmitted, N (%) P-valuea CHRND_MS and 83 were informative for TDT. Hetero- zygous single parents exhibited CHRND_MS genotypes 258 4 (4.8) 4 (4.8) reflecting allele frequencies observed in the control 260 0 (0) 7 (8.4) ¼ ¼ 262 32 (38.6) 29 (34.9) population, including 260/262 (n 3), 262/264 (n 1), 264 3 (3.6) 5 (6.0) 262/266 (n ¼ 10), 262/268 (n ¼ 1), 258/266 (n ¼ 2), 264/ 266 26 (31.3) 33 (39.8) 266 (n ¼ 1) and 266/268 (n ¼ 4). As shown in Table 1, the 268 16 (19.3) 3 (3.6) 0.0154 CHRND_MS*268 allele was transmitted to 16 children 270 2 (2.4) 2 (2.4) and untransmitted in three cases (Pc ¼ 0.0154). In contrast, transmission of rs2767 alleles was balanced aExact probabilities were calculated using the binomial distribution. (17 transmitted vs 16 untransmitted), whereas rs1004432 They were corrected by multiplication by the number of alleles and rs155093 were not polymorphic in our population tested, including six alleles for the microsatellite (the allele 270 sample. Two-locus TDT disclosed a preferential trans- cannot yield significant results) and one allele for the rs2767 SNP. mission of the MS_268 allele combined to the ‘C’ allele of The only P-value o0.05 reported corresponded to a nominal P- rs2767, with a borderline significance (eight transmis- value of 0.0022. sions vs one nontransmission, corrected P ¼ 0.056 for four haplotypes tested). Altogether, the data demonstrated linkage of CHRND to MG, with a positive association of Table 2 CHRND_MS phenotype frequencies in thymoma and CHRND_MS*268. nonthymoma MG patients and in controls A case–control study comparing 444 unrelated Cauca- soid patients and 168 healthy control subjects was then Allele Nonthymoma Thymoma Controls performed (Table 2). The patient group included the 114 probands of the family-based analysis and 330 additional n ¼ 350 % n ¼ 84 % n ¼ 168 % unrelated patients. They were included in the study with their written informed consent and in keeping with the 258 14 4 6 7.1 3 1.8 tenets of the Declaration of Helsinki. They fulfilled 260 13 3.7 7 8.3 3 1.8 clinical and electromyographic diagnostic criteria of 262 274 78.3 75 89.3 136 81.0 acquired generalized MG. Moreover, as the CHRND 264 8 2.3 2 2.4 11 6.5 gene was a candidate, all the patients had confirmed 266 196 56 37 44 92 54.8 268 52 14.9a 8 9.5 15 8.9 positive titers of anti-AChR autoantibodies (40.6 nM), 270 7 2 2 2.4 4 2.4 determined with a radioimmunoprecipitation assay.22 In 282 patients who underwent thymectomy, histopathol- aOdds ratio for the comparison with controls: 1.78 (P ¼ 0.038). See ogy of the thymus was assessed as described elsewhere.7 details in Table 3. Patients with a thymoma were considered as a separate group. As a whole, they present a different clinical profile, often with severe clinical symptoms and they require specific treatments.1–3 In addition, in contrast to French blood donors recruited at the Necker Hospital nonthymoma patients, they showed no genetic associa- and 57 unrelated Caucasian French healthy individuals tion, whether at the HLA complex7 or at the CHRNA1 obtained from the Fondation Jean Dausset – CEPH locus (Giraud et al, submitted for publication). The (Centre d’Etudes du Polymorphisme Humain, Paris). control groups included 111 volunteering Caucasian Both groups showed very similar allele frequencies

Genes and Immunity CHRND and myasthenia gravis M Giraud et al 82 Table 3 Odds ratios for CHRND_MS*268 in subgroups of nonthymoma MG patients compared to controls

Patients N MS*268(+) % OR (95% CI) P-value

Thymoma cases 84 8 9.5 1.07 All nonthymoma cases 350 52 14.9 1.78 (1.037 À +N) 0.038 TDT probands 94 17 18.1 2.24 (1.12 À +N) 0.026 Additional cases 256 35 13.7 1.61 Anti-titin antibody tested 294 44 15.0 1.79 (1.03 À +N) 0.04 Anti-titin positive 93 10 10.8 1.23 Anti-titin negative 201 34 16.9 2.07 (1.16 À +N) 0.017

aOdds ratios (OR), their 95% confidence interval (CI) and the P-values were calculated exactly with the Logxact software (Cytel corp, Cambridge, MA, USA).

(Supplemental Table) and were therefore pooled not those positive, now showed a significant association (Table 2). with the allele 268, with an odds ratio of 2.07 (P ¼ 0.017). As shown in Table 2, nonthymoma patients showed a Other alleles were similarly distributed (Supplemental modest but significant increase in the frequency of Table). Altogether, the data indicated that the association CHRND_MS*268, compared with healthy control sub- of the CHRND_MS*268 allele was restricted to the subset jects (14.9 vs 8.9%, OR ¼ 1.78, P ¼ 0.038). Owing to the of MG patients lacking anti-titin antibodies and con- prior knowledge of an association of this allele, the P- firmed that patients expressing these antibodies form a value was tested one-sided and was not corrected for the distinct subset, both clinically and genetically. number of alleles. The frequencies of other alleles, Dinucleotide repeats may adopt an unusual conforma- inferred from phenotype frequencies, were similar to tion that may alter gene expression, as was recently those, transmitted or untransmitted, observed in the TDT demonstrated for a CA repeat in the nucleolin gene.23,24 analysis (Table 1). In contrast, patients with thymoma However, given the complex properties of linkage showed no association with the allele 268, providing disequilibrium, a thorough analysis of the polymorph- further support for thymoma patients being a pathogen- ism of CHRND and of neighbor loci along with etically distinct subset of MG patients. functional studies will be necessary to establish the The group of nonthymoma patients by itself was also CHRND_MS microsatellite as a causative polymorphism heterogeneous. Probands of the family-based analysis in MG predisposition. were significantly younger at onset of disease than the The CHRNG gene that encodes the g-subunit of AChR additional cases (mean7s.d.: 29.2711.6 vs 46.6720.7 is situated at 4.5 kb on the telomeric side of CHRND years, Po1 Â10À12). The former were more significantly (Figure 1). Two SNPs, rs1190434 and rs1656389, both associated with the CHRND_MS*268 allele than the localized in the fifth intron of CHRNG and separated by whole group, whereas the latter showed no association 13 bp, were selected among five available in dbSNP (Table 3). Other alleles were not distributed differently (build 96) and were typed in the 444 patients. Not between the two subsets (Supplemental Table). The age surprisingly, pairwise linkage disequilibria between the at onset of disease was not the actual discriminating various loci were highly significant (Po1 Â10À6). How- parameter as the presence of the CHRND_MS*268 allele ever, the linkage disequilibrium D0 measure25,26 between did not influence the age at onset (nonparametric Mann– CHRND_MS and rs2767 (the CHRND SNP), and that Whitney test comparing nonthymoma patients positive between the two CHRNG SNPs were close to the unity, and those negative for the allele 268, not significant). We in marked contrast with the values of D0 for pairwise and others have previously shown that, among non- associations between CHRND and CHRNG polymorph- thymoma patients, patients expressing anti-titin antibo- isms (Figure 1). These data suggested that although dies in their serum formed a characteristic subset with a CHRND and CHRNG were closely linked, historical late onset of disease and with intermediate titers of anti- recombinations or evolutionary forces had individua- AChR antibodies.22 Moreover, these patients showed lized the two genes. Finally, a TDT with the CHRNG specific HLA associations, positive with DR7 and SNPs was performed in the sample of 114 one-generation negative with DR3.7 Anti-titin antibodies had been families. Transmission of alleles of both SNPs was assayed in 294 patients out of the 350 nonthymoma balanced (data not shown). patients. Seven out of the 57 TDT probands (12.3%) and In conclusion, our study demonstrates linkage and 86 out of 237 additional cases (36.3%) who were tested association of the CHRND locus with MG. It will be displayed serum anti-titin antibodies. As previously essential to investigate different patient samples to described, patients with anti-titin antibodies had a much obtain confirmation of the results. The data nevertheless later onset of disease (63712 years) than those without warrant a detailed characterization of CHRND and the antibodies (34.9717.2 years, Po1 Â10À16). The whole CHRNG gene polymorphisms in MG patients. They also sample showed frequencies for CHRND_MS alleles strengthen the interest for the genes encoding self- similar to those of the whole group of nonthymoma antigens in the analyses of a genetic predisposition to patients (Supplemental Table). As shown in Table 3, there autoimmune diseases. was a modest but significant increase of the allele 268. Supplementary information accompanies the paper on Moreover, patients negative for anti-titin antibodies, but Gene and Immunity (http://www.nature.com/gene)

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