P2RY8 plays essential roles in human B cell tolerance and prevents lupus

Samples were obtained from Renji Hospital Biobank, Shanghai Jiaotong University School of medicine (Ethical approval number: [2013] 126). All donors signed the informed consent. Genomic DNA was isolated from blood using QIAamp DNA Blood kit (QIAGEN) and was sequenced by Illumina platform (Novogene, Beijing). Bioinformatic analysis were performed at China-Australia Centre for Personalised Immunology (CACPI). Relatedness was checked using Peddy45. Identified variants were scored based upon a predesigned algorithm incorporating frequency of SNP, potential consequence of resulting amino acid changes, in silico predictions of damage (Polyphen, SIFT, and CADD), and function of domains bearing mutation. All variants of interest in P2RY8 were further confirmed by Sanger sequencing. Whole exome sequencing (WES) of 61 trios in which the proband had childhood-onset SLE identified a novel heterozygous missense P2RY8 variant L257F (c.769C>T) (Fig.1a and Extended Data Fig. 1a). The girl suffered from lupus, glomerulonephritis (histological class V), iron-deficiency anemia, and multiple bone infarcts affecting knees, femur, tibia, and fibula. Sanger sequencing of both parents coupled with relatedness check established that the L257F variant occurred de novo (it was not present in either parent) (Fig.1c and Extended Data Fig. 1b,c). WES of an additional cohort of 118 Chinese SLE patients identified (Fig.1a) an additional rare heterozygous P2RY8 variant E323G (c.968A>G) in two adult-onset SLE patients, which was Sanger-validated (Fig.1d). Both P2RY8 L257F and E323G variants were predicted to be pathogenic according to SIFT and CADD score, and the L257F variant was also predicted to be damaging by polyphen (Fig.1b). Variant frequency databases including Exome Aggregation Consortium (ExAC) and the Genome Aggregation Database (gnomAD) indicated the L257F variant was novel, and the E323G variant minor allele frequency was less than 0.3% (ExAC = 0.00021) (Fig.1b). The L257F variant occurs in a highly conserved transmembrane region (Fig.1e,f). The E323 site is located in the C-terminus, and is also conserved in multiple species (Fig.1e,f).