each exon of siae was amplified from genomic dna from individual subjects and subjected to automated sequencing . residue numbering was based on enst00000263593 ( ensembl ) , corresponding to the genbank accession number np_733746 . genomic dna was extracted from clotted blood specimens from patients with autoimmune disease using a qiaamp dna blood mini kit ( qiagen , valencia , ca ) according to the manufacturer s instructions . all 10 exons of the human siae gene were amplified by pcr using intronic primers ( supplementary table s4 ) . purification of the amplified products , bidirectional automated sequencing and sequence analysis were performed as described earlier13 . dnas from controls were obtained from ebv - immortalized lymphoblastoid cell - lines established from healthy blood donors14 . all blood samples were collected with approval from the mgh / partners human studies institutional review board and from the institutional review board at north shore long island jewish health system . a human siae cdna was cloned from mda - mb 231 cells and a full length flag - tagged human siae expression construct ( in pcdna3.1 ) was generated . this clone was targeted for mutagenesis using pfuturbo dna polymerase ( stratagene , ca , usa ) . each siae variant ( other than the w48x truncation ) was recreated by site directed mutagenesis as a c - terminal flag tagged human siae cdna in an expression vector site directed mutagenesis was used to create the s127a variant with a defect in the catalytic site as well as each of the variants listed in table i. the pcr products were digested overnight with dpni ( 10 units ; stratagene ) and transformed into top10 chemically competent cells ( invitrogen , carlsbad , ca ) . lysates and supernatants were immunoprecipitated with anti - flag antibodies and catalytic activity of the immunoprecipitated esterase was assayed by a fluorimetric method15 . equivalent amounts of each lysate and supernatant were immunoprecipitated for the catalytic assay as well as for quantitation of the flag - tagged protein by an immunoblot assay on the li - cor odyssey , using a mouse monoclonal anti - flag antibody ( sigma ) and an ir dye 800cw labeled goat anti - mouse igg ( li - cor ) as a secondary antibody . immunoprecipitation , metabolic labeling , and pulse chase studies were performed as described in ref.1 . these assays were carried out by co - transfecting cdnas encoding v5-tagged wild type murine siae , together with flag - tagged murine versions of siae mutants discovered in subjects with rheumatoid arthritis into 293 t cells . the v5-tagged wild type proteins in cell lysates were immunoprecipitated using mouse monoclonal anti - v5 antibody ( invitrogen ) , for quantitative immunoblot and esterase activity assays . expression of flag - tagged mutants was also monitored by immunoprecipitation and western blot assays . the method used was described in murine lymphocytes in reference1and is based on the method described originally by krishna and varki16 . briefly human b cells were stained with antibodies to cd19 and cd27 ( bd pharmingen ) and incubated either with or without f(ab)2 , polyclonal rabbit anti - human igm ( dako ) . cells were also stained either with fitc - f(ab)2 , goat anti - human igg , fc specific ( jackson immunoresearch ) alone , or the che - fcd reagent ( an influenza c hemagglutin esterase - fused to the fc portion of human igg , chemically treated with diisopropyl fluorophosphate ) complexed with fitc - f(ab)2 fragment goat anti - human igg , fc specific . each exon of siae was amplified from genomic dna from individual subjects and subjected to automated sequencing . residue numbering was based on enst00000263593 ( ensembl ) , corresponding to the genbank accession number np_733746 . genomic dna was extracted from clotted blood specimens from patients with autoimmune disease using a qiaamp dna blood mini kit ( qiagen , valencia , ca ) according to the manufacturer s instructions . all 10 exons of the human siae gene were amplified by pcr using intronic primers ( supplementary table s4 ) . purification of the amplified products , bidirectional automated sequencing and sequence analysis were performed as described earlier13 . dnas from controls were obtained from ebv - immortalized lymphoblastoid cell - lines established from healthy blood donors14 . all blood samples were collected with approval from the mgh / partners human studies institutional review board and from the institutional review board at north shore long island jewish health system . a human siae cdna was cloned from mda - mb 231 cells and a full length flag - tagged human siae expression construct ( in pcdna3.1 ) was generated . this clone was targeted for mutagenesis using pfuturbo dna polymerase ( stratagene , ca , usa ) . each siae variant ( other than the w48x truncation ) was recreated by site directed mutagenesis as a c - terminal flag tagged human siae cdna in an expression vector site directed mutagenesis was used to create the s127a variant with a defect in the catalytic site as well as each of the variants listed in table i. the pcr products were digested overnight with dpni ( 10 units ; stratagene ) and transformed into top10 chemically competent cells ( invitrogen , carlsbad , ca ) . lysates and supernatants were immunoprecipitated with anti - flag antibodies and catalytic activity of the immunoprecipitated esterase was assayed by a fluorimetric method15 . equivalent amounts of each lysate and supernatant were immunoprecipitated for the catalytic assay as well as for quantitation of the flag - tagged protein by an immunoblot assay on the li - cor odyssey , using a mouse monoclonal anti - flag antibody ( sigma ) and an ir dye 800cw labeled goat anti - mouse igg ( li - cor ) as a secondary antibody . immunoprecipitation , metabolic labeling , and pulse chase studies were performed as described in ref.1 . these assays were carried out by co - transfecting cdnas encoding v5-tagged wild type murine siae , together with flag - tagged murine versions of siae mutants discovered in subjects with rheumatoid arthritis into 293 t cells . the v5-tagged wild type proteins in cell lysates were immunoprecipitated using mouse monoclonal anti - v5 antibody ( invitrogen ) , for quantitative immunoblot and esterase activity assays . expression of flag - tagged mutants was also monitored by immunoprecipitation and western blot assays . the method used was described in murine lymphocytes in reference1and is based on the method described originally by krishna and varki16 . briefly human b cells were stained with antibodies to cd19 and cd27 ( bd pharmingen ) and incubated either with or without f(ab)2 , polyclonal rabbit anti - human igm ( dako ) . cells were also stained either with fitc - f(ab)2 , goat anti - human igg , fc specific ( jackson immunoresearch ) alone , or the che - fcd reagent ( an influenza c hemagglutin esterase - fused to the fc portion of human igg , chemically treated with diisopropyl fluorophosphate ) complexed with fitc - f(ab)2 fragment goat anti - human igg , fc specific .
sialic acid acetylesterase ( siae ) is an enzyme that negatively regulates b lymphocyte antigen receptor signaling and is required for the maintenance of immunological tolerance in mice1 , 2 . 
 heterozygous loss - of - function germline rare variants and a homozygous defective polymorphic variant of siae were identified in 24/923 caucasian subjects with relatively common autoimmune disorders and in 2/648 caucasian controls . 
 all heterozygous loss - of - function siae mutations tested were capable of functioning in a dominant negative manner . 
 a homozygous secretion - defective polymorphic variant of siae was catalytically active , lacked the ability to function in a dominant negative manner , and was seen in 8 autoimmune subjects but in no control subjects . 
 the odds ratio for inheriting defective siae alleles was 8.6 in all autoimmune subjects , 8.3 in subjects with rheumatoid arthritis , and 7.9 in subjects with type i diabetes . 
 functionally defective siae rare and polymorphic variants represent a strong genetic link to susceptibility in relatively common human autoimmune disorders .