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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

CHiP-qtPCR showing binding of NFκB p50 as well as NFκBp65 within the genomic segment contained in the 1.2 Kb haplotype window in Daudi cells. (A) mRNA expression of the BLK gene after BcR stimulation. (B) qtPCR of the portion containing SNP rs2248932 within the 1.2 kb genomic segment in intron 1 of human BLK. (C) qtPCR of the IkBa gene used as positive control.
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Figure 3: CHiP-qtPCR showing binding of NFκB p50 as well as NFκBp65 within the genomic segment contained in the 1.2 Kb haplotype window in Daudi cells. (A) mRNA expression of the BLK gene after BcR stimulation. (B) qtPCR of the portion containing SNP rs2248932 within the 1.2 kb genomic segment in intron 1 of human BLK. (C) qtPCR of the IkBa gene used as positive control.

Mentions: We first attempted the identification of functional regulatory variants across the promoter blocks B1 and B3. Bioinformatic analysis revealed that B1 was significantly enriched for H3K4me3 sites, a hallmark of active promoters (figure 1, online figure S2 and online table S3). Furthermore, SNPs in B1 and B3, when compared with randomly chosen nucleotides, were enriched for binding sites for the transcription factor NFκBp65 and IRF4 but not PAX5 (using data from ENCODE project Chip-Seq for lymphoblastoid cell lines), while SNPs in the non-associated blocks B2 and B4 were not (figure 1, online figure S2 and online table S3). However, we were unable to single out a unique variant to explain the correlation between low BLK gene expression and risk alleles of SNPs in B1 and B3 due to the strong LD. We hypothesised that multiple sites would therefore explain a coordinated regulatory effect on the expression of BLK. Nevertheless, the 1.2 Kb haplotype window with the strongest genetic association and within B3 contained SNP rs2248932 that overlapped with a DNAse hypersensitive cluster and with a strong NFκB binding site (online figure S3 and online table S4). To confirm if NFκB binds to this segment of BLK we performed a CHiP-qtPCR experiment in a human Daudi cell line (figure 3A–C). We observed that NFκBp50 binds somewhat earlier than NFκBp65 (30 min post-BcR stimulation). The IkBa promoter served as control (figure 3C). Simultaneously, expression of BLK is reduced after activation (figure 3A).


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)

CHiP-qtPCR showing binding of NFκB p50 as well as NFκBp65 within the genomic segment contained in the 1.2 Kb haplotype window in Daudi cells. (A) mRNA expression of the BLK gene after BcR stimulation. (B) qtPCR of the portion containing SNP rs2248932 within the 1.2 kb genomic segment in intron 1 of human BLK. (C) qtPCR of the IkBa gene used as positive control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: CHiP-qtPCR showing binding of NFκB p50 as well as NFκBp65 within the genomic segment contained in the 1.2 Kb haplotype window in Daudi cells. (A) mRNA expression of the BLK gene after BcR stimulation. (B) qtPCR of the portion containing SNP rs2248932 within the 1.2 kb genomic segment in intron 1 of human BLK. (C) qtPCR of the IkBa gene used as positive control.
Mentions: We first attempted the identification of functional regulatory variants across the promoter blocks B1 and B3. Bioinformatic analysis revealed that B1 was significantly enriched for H3K4me3 sites, a hallmark of active promoters (figure 1, online figure S2 and online table S3). Furthermore, SNPs in B1 and B3, when compared with randomly chosen nucleotides, were enriched for binding sites for the transcription factor NFκBp65 and IRF4 but not PAX5 (using data from ENCODE project Chip-Seq for lymphoblastoid cell lines), while SNPs in the non-associated blocks B2 and B4 were not (figure 1, online figure S2 and online table S3). However, we were unable to single out a unique variant to explain the correlation between low BLK gene expression and risk alleles of SNPs in B1 and B3 due to the strong LD. We hypothesised that multiple sites would therefore explain a coordinated regulatory effect on the expression of BLK. Nevertheless, the 1.2 Kb haplotype window with the strongest genetic association and within B3 contained SNP rs2248932 that overlapped with a DNAse hypersensitive cluster and with a strong NFκB binding site (online figure S3 and online table S4). To confirm if NFκB binds to this segment of BLK we performed a CHiP-qtPCR experiment in a human Daudi cell line (figure 3A–C). We observed that NFκBp50 binds somewhat earlier than NFκBp65 (30 min post-BcR stimulation). The IkBa promoter served as control (figure 3C). Simultaneously, expression of BLK is reduced after activation (figure 3A).

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