Although genome-wide linkage analysis of IgAN has revealed several susceptibility loci, the causative genes have not been identified. From the point of view of genetic heterogeneity of familial IgAN, an oligo/polygenic and multiple susceptibility gene model for the disease has been proposed. Recently, exome learn more sequencing has emerged as a powerful and cost-effective strategy for dissecting the genetic basis of diseases. Methods: To identify the genetic causality of familial IgAN,

we applied exome sequencing to a family comprising four biopsy-proven IgAN patients clustered in a dominant transmission mode. The whole exomes of four affected, two unmanifested carriers, and two unaffected individuals were captured and subjected to massive parallel sequencing. Variants identified by exome sequencing were filtered on the basis of variant annotation, functional expectation, and allele frequency. The affected individuals in the family were expected to share the same causal variant. Genome-wide linkage analysis was concurrently

performed for the family using the high-throughput linkage analysis system SNP HiTLink. Sequence analysis of the EEA1 gene was performed in other members of the family and in 27 additional cases with IgAN. The Human Genetic Variation database was used as a reference for the exome sequence data of the Japanese population. Results: Several filtering procedures for extracting candidates with disease-causing variants were effectively used as follows. The first step involved performing variant annotation on the basis of dbSNP FDA approved Drug Library cell assay entries, 1000 Genome O-methylated flavonoid Project, and amino acid substitutions to retain novel nonsynonymous variants. The next filtering

stage was performed on the basis of allele frequency, and an interval of 30%–70% was used as the cut-off threshold. Finally, 13 variants that were shared only by the affected individuals in the family were selected as candidate genes for familial IgAN. Linkage analysis of the family revealed linkage signals at nine loci. Among the candidates, a novel missense variant F161Y in EEA1 that encodes early endosome antigen 1 (a Rab5 effector protein that facilitates the docking and tethering of incoming endocytic vesicles) was located within a linkage locus with a maximum LOD score of 1.68. Furthermore, the F161Y variant completely cosegregated in the family, and this variant is present in a highly conserved region across zebrafish to human. Sequence analysis of EEA1 revealed that among the additional 27 familial IgAN cases, six families carried three other variants (R1262W, N1072K, and E1010G) within EEA1 with reduced penetrance. The frequencies of these EEA1 variants in familial IgAN were significantly higher than those in the Human Genetic Variation database.