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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.

Delgado-Vega AM, Dozmorov MG, Quirós MB, Wu YY, Martínez-García B, Kozyrev SV, Frostegård J, Truedsson L, de Ramón E, González-Escribano MF, Ortego-Centeno N, Pons-Estel BA, D'Alfonso S, Sebastiani GD, Witte T, Lauwerys BR, Endreffy E, Kovács L, Vasconcelos C, da Silva BM, Wren JD, Martin J, Castillejo-López C, Alarcón-Riquelme ME - Ann. Rheum. Dis. (2012)

Bottom Line: 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.The 71Thr decreased BLK protein half-life.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, 751 85, Sweden.

ABSTRACT

Objectives: 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.

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Related in: MedlinePlus

Reduced half-life of BLK protein in the presence of the 71Thr mutation. (A) Plasmids expressing each of the two BLK isoforms fused with V5 were transfected into HEK293T cells. Twenty-four hours after transfection, the cells were treated with 20 µg/ml cycloheximide (CHX) for various times as indicated. The recombinant proteins were detected by western blot using anti-V5 antibody. Tubulin was used as loading control. (B) Quantification of the blots from three independent experiments; the SD at each time point is represented by bars. After 8 h of inhibition of protein synthesis, the level of BLK 71Thr was reduced to half while the wild type isoform Ala71 remained highly stable.
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Figure 5: Reduced half-life of BLK protein in the presence of the 71Thr mutation. (A) Plasmids expressing each of the two BLK isoforms fused with V5 were transfected into HEK293T cells. Twenty-four hours after transfection, the cells were treated with 20 µg/ml cycloheximide (CHX) for various times as indicated. The recombinant proteins were detected by western blot using anti-V5 antibody. Tubulin was used as loading control. (B) Quantification of the blots from three independent experiments; the SD at each time point is represented by bars. After 8 h of inhibition of protein synthesis, the level of BLK 71Thr was reduced to half while the wild type isoform Ala71 remained highly stable.

Mentions: At the translational level, the substitution of alanine for threonine implies the formation of a putative phosphorylation site that could lead to the reduction of the half-life of the protein. We investigated if this was the case. Using constructs containing each allele of rs55758736 in transfection experiments we observed that, indeed, 71Thr had an accelerated degradation after treatment with the protein synthesis inhibitor cycloheximide as compared with the Ala71 allele (figure 5 A,B and figure S4).


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.

Delgado-Vega AM, Dozmorov MG, Quirós MB, Wu YY, Martínez-García B, Kozyrev SV, Frostegård J, Truedsson L, de Ramón E, González-Escribano MF, Ortego-Centeno N, Pons-Estel BA, D'Alfonso S, Sebastiani GD, Witte T, Lauwerys BR, Endreffy E, Kovács L, Vasconcelos C, da Silva BM, Wren JD, Martin J, Castillejo-López C, Alarcón-Riquelme ME - Ann. Rheum. Dis. (2012)

Reduced half-life of BLK protein in the presence of the 71Thr mutation. (A) Plasmids expressing each of the two BLK isoforms fused with V5 were transfected into HEK293T cells. Twenty-four hours after transfection, the cells were treated with 20 µg/ml cycloheximide (CHX) for various times as indicated. The recombinant proteins were detected by western blot using anti-V5 antibody. Tubulin was used as loading control. (B) Quantification of the blots from three independent experiments; the SD at each time point is represented by bars. After 8 h of inhibition of protein synthesis, the level of BLK 71Thr was reduced to half while the wild type isoform Ala71 remained highly stable.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3375585&req=5

Figure 5: Reduced half-life of BLK protein in the presence of the 71Thr mutation. (A) Plasmids expressing each of the two BLK isoforms fused with V5 were transfected into HEK293T cells. Twenty-four hours after transfection, the cells were treated with 20 µg/ml cycloheximide (CHX) for various times as indicated. The recombinant proteins were detected by western blot using anti-V5 antibody. Tubulin was used as loading control. (B) Quantification of the blots from three independent experiments; the SD at each time point is represented by bars. After 8 h of inhibition of protein synthesis, the level of BLK 71Thr was reduced to half while the wild type isoform Ala71 remained highly stable.
Mentions: At the translational level, the substitution of alanine for threonine implies the formation of a putative phosphorylation site that could lead to the reduction of the half-life of the protein. We investigated if this was the case. Using constructs containing each allele of rs55758736 in transfection experiments we observed that, indeed, 71Thr had an accelerated degradation after treatment with the protein synthesis inhibitor cycloheximide as compared with the Ala71 allele (figure 5 A,B and figure S4).

Bottom Line: 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.The 71Thr decreased BLK protein half-life.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, 751 85, Sweden.

ABSTRACT

Objectives: 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.

Show MeSH
Related in: MedlinePlus