In the test stage, we identified 142 rare variants in exon 5, of 54 different types with MAF < 1% (Figure 1B, Table 2). Seven of the 12 types of rare variants identified in the discovery stage in exon 5 were observed again in the test stage. Of the 54 unique rare variants, 35 were observed only once, and eight were observed more than five times. Most of the rare variants (64.8% of the 54 types of rare variants, and 83.1% of the total variants) were located in the region encoding the cytoplasmic loop of the α4 subunit. Twenty-four of the 54 unique rare variants were nonsynonymous variants, which were all missense, with 18 of them located in the region encoding the cytoplasmic loop. Of these cytoplasmic missense rare variants, 30 were found in comparison subjects, and 14 were found in cases (Figure 2). Comparison subjects had a significantly higher frequency of rare nonsynonymous variants in the cytoplasmic loop region than the cases (FET p=0.009; association test p=0.009, OR=0.43, 95%CI=0.23-0.81; WSM p=0.014). The frequency of rare nonsynonymous variants located in
