All patients were of self-reported white ancestry. Serum samples were available for 82 patients; anti-PLA2R antibodies Bay K 8644 Bay K 8644 were present in 43 samples. mutations can be the cause of a signal identified by GWAS.8 Although iMN is not a simple genetic disorder with classic Mendelian inheritance, the strong and significant association of this phenotype to (and the locus) leads to the hypothesis that rare genetic variants Bay K 8644 within the coding region of the gene may explain antibody formation. In the current study, we performed sequencing of the coding regions of the gene to confirm this hypothesis. Results Patient Characteristics Ninety-five patients with biopsy-proven iMN were included: 48 from the French cohort and 47 from the Dutch cohort, respectively. Patients were mostly male (75%), and the mean age SD at diagnosis was 5115 years. All patients were of self-reported white ancestry. Serum samples were available for 82 patients; anti-PLA2R antibodies were present in 43 samples. In addition, 17 patients were considered positive according to the presence of PLA2R antigen in their kidney biopsy specimen (Supplemental Table S1). Sequencing All 30 exons, including essential splice sites (defined as the two intronic nucleotidescanonical GT and AGat the intron-exon boundaries, respectively) were sequenced by Sanger technology. We identified 18 variants, including 3 in noncoding regions (Table 1). Table 1. Sequence variants in observed in the present study Bay K 8644 (95 patients with iMN) Valuevalue, comparison of variation allele frequencies in controls and patients; ref cDNA, reference allele cDNA (given that is around the unfavorable strand); cDNA, “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_007366.4″,”term_id”:”307548854″,”term_text”:”NM_007366.4″NM_007366.4; prot level, “type”:”entrez-protein”,”attrs”:”text”:”NP_031392.3″,”term_id”:”55953104″,”term_text”:”NP_031392.3″NP_031392.3; Ess, essential splice site; ss, splice site; aa, amino acid; WMGL, motif; FNII, fibronectin type II domain name; CRD, cysteine-rich domain name; UTR, untranslated region. aRare variants. bSignificantly associated with iMN. cNovel variants. dNot reported in 1000 Genomes; allele frequency from Single Nucleotide Polymorphism Database, release 136. Of the 18 variants observed, 9 were rare or novel (see the Concise Methods section for definitions). These 9 variants were all encountered in a heterozygous state in one patient each, except for the rare variant rs149133741 (c.2038G T, p.Val680Leu), which was observed in two patients. Seven of these rare variants (rs141800672, rs149256089, rs150221555, rs140427239, rs149960520, rs149133741, and rs181959329) have been reported in publicly available data sets with very low minor allele frequencies (0.2%). We identified two novel missense variants that have not been previously reported, which result in amino acid changes (c.1160G A, p.Arg387His; c.2060T C, p.Leu687Pro). The novel variant c.1160G A, p.Arg387His and the splice site change c.3850+1G A (IVS26+1G A) (rs181959329) were carried by the same patient. Additionally, nine common SNPs were observed; six of them in the coding regions (rs4665143, rs3749117, rs35771982, rs33985939, rs72954858, and rs3828323) and three in noncoding regions (rs2715918, rs925409, and rs3749119). In the subgroup of 60 anti-PLA2R-positive patients, four of Mouse Monoclonal to C-Myc tag the rare variants (rs149256089, rs140427239, rs149960520, and rs149133741) were each observed once in four different patients. The two novel variants were not observed in this subgroup (Table 2). Table 2. Sequence variants in observed in 60 anti-PLA2R1Cpositive patients Valuevalue, comparison of variation allele frequencies in controls and patients; ref cDNA, reference allele cDNA (given that is around the unfavorable strand); cDNA, “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_007366.4″,”term_id”:”307548854″,”term_text”:”NM_007366.4″NM_007366.4; prot level, “type”:”entrez-protein”,”attrs”:”text”:”NP_031392.3″,”term_id”:”55953104″,”term_text”:”NP_031392.3″NP_031392.3; aa, amino acid; WMGL, motif; FNII, fibronectin type II domain name; CRD, cysteine-rich domain name; UTR, untranslated region. aSignificantly associated with iMN. bRare variants. cNot reported in 1000 Genomes; allele frequency from Single Nucleotide Polymorphism Database, release 136. Of the 15 observed coding variants, 12 (rs149133741, rs149960520, rs140427239, rs33985939, rs150221555, rs35771982, rs3749117, rs149256089, rs141800672, rs4665143, and the novel p.Arg387His and p.Leu687Pro) are located within the regions coding for known domains of PLA2R1, of which 1 (rs3749117) is located within the WMGL motif of the C-type lectin-like domain name (CTLD)-1 of PLA2R1. Two other coding variants, one common (rs33985939, p.Arg404His) and one rare (rs140427239, p.Tyr499Cys), are situated one and two amino acids, respectively, away from the cysteine residues involved in disulfide bond formation (C4 and C1 in CTLD2; see Table 1 and Figure 1). Open in a separate window Figure 1. Structure of PLA2R1. The amino acid sequence of PLA2R1 starts with a.