Fine mapping and conditional analysis identify a new mutation in the autoimmunity susceptibility gene BLK that leads to reduced half-life of the BLK protein.
OBJECTIVE
To perform fine mapping of the autoimmunity susceptibility gene BLK and identify functional variants involved in systemic lupus erythematosus (SLE).


METHODS
Genotyping of 1163 European SLE patients and 1482 controls and imputation were performed covering the BLK gene with 158 single-nucleotide polymorphisms. Logistic regression analysis was done using PLINK and conditional analyses using GENABEL's test score. Transfections of BLK constructs on HEK293 cells containing the novel mutation or the wild type form were analysed for their effect on protein half-life using a protein stability assay, cycloheximide and western blot. CHiP-qPCR for detection of nuclear factor κ B (NFkB) binding.


RESULTS
Fine mapping of BLK identified two independent genetic effects with functional consequences: one represented by two tightly linked associated haplotype blocks significantly enriched for NFκB-binding sites and numerous putative regulatory variants whose risk alleles correlated with low BLK mRNA levels. Binding of NFkBp50 and p65 to an associated 1.2 Kb haplotype segment was confirmed. A second independent genetic effect was represented by an Ala71Thr, low-frequency missense substitution with an OR=2.31 (95% CI 1.38 to 3.86). The 71Thr decreased BLK protein half-life.


CONCLUSIONS
These results show that rare and common regulatory variants in BLK are involved in disease susceptibility and both, albeit independently, lead to reduced levels of BLK protein.
Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, 751 85, Sweden.
Angélica M
AM
Delgado-Vega
Mikhail G
MG
Dozmorov
Manuel Bernal
MB
Quirós
Ying-Yu
YY
Wu
Belén
B
Martínez-García
Sergey V
SV
Kozyrev
Johan
J
Frostegård
Lennart
L
Truedsson
Enrique
E
de Ramón
María F
MF
González-Escribano
Norberto
N
Ortego-Centeno
Bernardo A
BA
Pons-Estel
Sandra
S
D'Alfonso
Gian Domenico
GD
Sebastiani
Torsten
T
Witte
Bernard R
BR
Lauwerys
Emoke
E
Endreffy
László
L
Kovács
Carlos
C
Vasconcelos
Berta Martins
BM
da Silva
Jonathan D
JD
Wren
Javier
J
Martin
Casimiro
C
Castillejo-López
Marta E
ME
Alarcón-Riquelme
D015415:Biomarkers / Q000378:metabolism; D002874:Chromosome Mapping; D015971:Gene Expression Regulation, Enzymologic; D005819:Genetic Markers / Q000235:genetics; D020022:Genetic Predisposition to Disease; D005838:Genotype; D057809:HEK293 Cells; D006207:Half-Life; D006801:Humans; D008180:Lupus Erythematosus, Systemic / Q000235:genetics*; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D011485:Protein Binding; D055550:Protein Stability; D014162:Transfection; D019061:src-Family Kinases / Q000235:genetics* / Q000378:metabolism
D015415:Biomarkers; D005819:Genetic Markers; D016328:NF-kappa B; C082599:protein-tyrosine kinase p55(blk); D019061:src-Family Kinases