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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

Systemic lupus erythematosus-associated single-nucleotide polymorphisms (SNPs) and haplotype blocks and genomic features of the BLK locus. From top to bottom: –log10 of the p value corrected by false discovery rate for 158 SNPs. Below, the association for haplotype windows of two, three and four SNPs is displayed; the horizontal black line marks the threshold for Bonferroni correction (p=0.05/323 windows tested). Only SNP windows contained within block B1 (5′ upstream, exon 1 – UTR – and beginning of intron 1), B3 (end of intron 1) and B5 (exons 4, 5 and 6) were associated after correction. The exon/intron structure of BLK is displayed next and then NFkBp65 (blue), IRF4 (yellow) and PAX5 (green) Chip-Seq identified binding sites and promoter-associated histone marks (H3K4Me3) according to ENCODE project. Recombination rate (cM/Mb) is displayed at the bottom.
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Figure 1: Systemic lupus erythematosus-associated single-nucleotide polymorphisms (SNPs) and haplotype blocks and genomic features of the BLK locus. From top to bottom: –log10 of the p value corrected by false discovery rate for 158 SNPs. Below, the association for haplotype windows of two, three and four SNPs is displayed; the horizontal black line marks the threshold for Bonferroni correction (p=0.05/323 windows tested). Only SNP windows contained within block B1 (5′ upstream, exon 1 – UTR – and beginning of intron 1), B3 (end of intron 1) and B5 (exons 4, 5 and 6) were associated after correction. The exon/intron structure of BLK is displayed next and then NFkBp65 (blue), IRF4 (yellow) and PAX5 (green) Chip-Seq identified binding sites and promoter-associated histone marks (H3K4Me3) according to ENCODE project. Recombination rate (cM/Mb) is displayed at the bottom.

Mentions: A total of 158 SNPs with MAF>0.005 were tested for association in 1163 European patients with SLE and 1482 age- and country-of-origin-matched controls. A meta-analysis demonstrated that the strongest association signal was in the BLK promoter region (rs998683, PCMH-corrected=1.05×10−4 and OR 1.38 (1.21 to 1.58)) (online table S1). However, even after correction for multiple testing, multiple association signals across the gene remained significant. To refine the BLK association, we tested sliding windows, which narrowed it down to three blocks (denominated B1, B3 and B5 in figure 1). The best model of association was observed for a 1.2 kb haplotype window between SNPs rs2248932 and rs9329246 located at the end of BLK's first intron (p=1.58×10−10, omnibus haplotype test).


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)

Systemic lupus erythematosus-associated single-nucleotide polymorphisms (SNPs) and haplotype blocks and genomic features of the BLK locus. From top to bottom: –log10 of the p value corrected by false discovery rate for 158 SNPs. Below, the association for haplotype windows of two, three and four SNPs is displayed; the horizontal black line marks the threshold for Bonferroni correction (p=0.05/323 windows tested). Only SNP windows contained within block B1 (5′ upstream, exon 1 – UTR – and beginning of intron 1), B3 (end of intron 1) and B5 (exons 4, 5 and 6) were associated after correction. The exon/intron structure of BLK is displayed next and then NFkBp65 (blue), IRF4 (yellow) and PAX5 (green) Chip-Seq identified binding sites and promoter-associated histone marks (H3K4Me3) according to ENCODE project. Recombination rate (cM/Mb) is displayed at the bottom.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Systemic lupus erythematosus-associated single-nucleotide polymorphisms (SNPs) and haplotype blocks and genomic features of the BLK locus. From top to bottom: –log10 of the p value corrected by false discovery rate for 158 SNPs. Below, the association for haplotype windows of two, three and four SNPs is displayed; the horizontal black line marks the threshold for Bonferroni correction (p=0.05/323 windows tested). Only SNP windows contained within block B1 (5′ upstream, exon 1 – UTR – and beginning of intron 1), B3 (end of intron 1) and B5 (exons 4, 5 and 6) were associated after correction. The exon/intron structure of BLK is displayed next and then NFkBp65 (blue), IRF4 (yellow) and PAX5 (green) Chip-Seq identified binding sites and promoter-associated histone marks (H3K4Me3) according to ENCODE project. Recombination rate (cM/Mb) is displayed at the bottom.
Mentions: A total of 158 SNPs with MAF>0.005 were tested for association in 1163 European patients with SLE and 1482 age- and country-of-origin-matched controls. A meta-analysis demonstrated that the strongest association signal was in the BLK promoter region (rs998683, PCMH-corrected=1.05×10−4 and OR 1.38 (1.21 to 1.58)) (online table S1). However, even after correction for multiple testing, multiple association signals across the gene remained significant. To refine the BLK association, we tested sliding windows, which narrowed it down to three blocks (denominated B1, B3 and B5 in figure 1). The best model of association was observed for a 1.2 kb haplotype window between SNPs rs2248932 and rs9329246 located at the end of BLK's first intron (p=1.58×10−10, omnibus haplotype test).

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