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Monozygotic twins discordant for common variable immunodeficiency reveal impaired DNA demethylation during naïve-to-memory B-cell transition.

Rodríguez-Cortez VC, Del Pino-Molina L, Rodríguez-Ubreva J, Ciudad L, Gómez-Cabrero D, Company C, Urquiza JM, Tegnér J, Rodríguez-Gallego C, López-Granados E, Ballestar E - Nat Commun (2015)

Bottom Line: Common variable immunodeficiency (CVID), the most frequent primary immunodeficiency characterized by loss of B-cell function, depends partly on genetic defects, and epigenetic changes are thought to contribute to its aetiology.Individual analysis confirms hypermethylation of these genes.Our results not only indicate a role for epigenetic alterations in CVID but also identify relevant DNA methylation changes in B cells that could explain the clinical manifestations of CVID individuals.

View Article: PubMed Central - PubMed

Affiliation: Chromatin and Disease Group, Cancer Epigenetics and Biology Programme (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Avda Gran Via 199-203, 08908L'Hospitalet de Llobregat, Barcelona, Spain.

ABSTRACT
Common variable immunodeficiency (CVID), the most frequent primary immunodeficiency characterized by loss of B-cell function, depends partly on genetic defects, and epigenetic changes are thought to contribute to its aetiology. Here we perform a high-throughput DNA methylation analysis of this disorder using a pair of CVID-discordant MZ twins and show predominant gain of DNA methylation in CVID B cells with respect to those from the healthy sibling in critical B lymphocyte genes, such as PIK3CD, BCL2L1, RPS6KB2, TCF3 and KCNN4. Individual analysis confirms hypermethylation of these genes. Analysis in naive, unswitched and switched memory B cells in a CVID patient cohort shows impaired ability to demethylate and upregulate these genes in transitioning from naive to memory cells in CVID. Our results not only indicate a role for epigenetic alterations in CVID but also identify relevant DNA methylation changes in B cells that could explain the clinical manifestations of CVID individuals.

No MeSH data available.


Related in: MedlinePlus

Validation of DNA methylation results by bisulfite pyrosequencing.(a) Schemes depicting a selection of genes indicating the differentially methylated CpG site (red line) and its relative location with respect to the TSS (located with an arrow). Additional CpG sites are represented with vertical black lines. CpG islands are represented with a green bar. (b) Bisulfite pyrosequencing of selected genes, all of which are relevant to B-cell biology. In addition to CD19+ B lymphocytes, CD4+ T lymphocytes and CD14+ cells (monocytes) were analysed for all genes. Methylation levels are represented as percentages. Bisulfite sequencing was technical triplicates of each sibling of the MWZ twin pair. Error bars correspond to s.d. Student's t-test comparisons with a P value above 0.05 are considered with a nonsignificant difference, and are not presented (c) β-values obtained from the DNA methylation array. The B cells from CVID twin have higher levels of DNA methylation (Δβ≥0.1) in the analysed genes than those from his healthy sibling. Black filled circles and black filled triangles, respectively, indicate the three β-values for the control twin and the CVID twin. The black line indicates the average of the β-values in each condition.
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f2: Validation of DNA methylation results by bisulfite pyrosequencing.(a) Schemes depicting a selection of genes indicating the differentially methylated CpG site (red line) and its relative location with respect to the TSS (located with an arrow). Additional CpG sites are represented with vertical black lines. CpG islands are represented with a green bar. (b) Bisulfite pyrosequencing of selected genes, all of which are relevant to B-cell biology. In addition to CD19+ B lymphocytes, CD4+ T lymphocytes and CD14+ cells (monocytes) were analysed for all genes. Methylation levels are represented as percentages. Bisulfite sequencing was technical triplicates of each sibling of the MWZ twin pair. Error bars correspond to s.d. Student's t-test comparisons with a P value above 0.05 are considered with a nonsignificant difference, and are not presented (c) β-values obtained from the DNA methylation array. The B cells from CVID twin have higher levels of DNA methylation (Δβ≥0.1) in the analysed genes than those from his healthy sibling. Black filled circles and black filled triangles, respectively, indicate the three β-values for the control twin and the CVID twin. The black line indicates the average of the β-values in each condition.

Mentions: To confirm the results obtained from the DNA methylation arrays, we performed bisulfite pyrosequencing of a selection of the aforementioned genes (Table 1), hypermethylated in B cells isolated from the same pair of twins discordant for CVID. In brief, the selection of genes was carried out among those displaying the largest changes in DNA methylation between the CVID and healthy sibling, and also to cover different functions in B-cell biology: PIK3CD, RPS6KB2 encode proteins involved in the cell proliferation, cell growth and protein synthesis, that occur in response to BCR signalling; BCL2L1 encodes a protein with anti-apoptotic effects. TCF3 encodes a transcription factor essential in the establishment of B-cell identity. PTPRCAP encodes a protein necessary for proper phosphatase activity of CD45 during BCR signalling, and CORO1B, KCNN4 and KCNC4 encode proteins that are involved in Ca+2 signalling. We focused on looking at the same CpG sites for which significant changes had been identified (Fig. 2a). Despite the modest differences in DNA methylation measured by the initial high-throughput strategy, we confirmed by bisulfite pyrosequencing a robust increase in DNA methylation in the CVID sibling with respect to the healthy sibling (Fig. 2b), with very similar values to those obtained using bead arrays (Fig. 2c). We also tested the DNA methylation changes in CD4+ cells (T lymphocytes) and CD14+ cells (monocytes) from the same individuals (Fig. 2b). In contrast with the robust increase in the frequency of B cells from the CVID sibling with respect to the healthy sibling, we observed no differences or changes in the opposite direction in T cells and monocytes, highlighting the specificity of the observed CVID-associated hypermethylation of these genes to B cells.


Monozygotic twins discordant for common variable immunodeficiency reveal impaired DNA demethylation during naïve-to-memory B-cell transition.

Rodríguez-Cortez VC, Del Pino-Molina L, Rodríguez-Ubreva J, Ciudad L, Gómez-Cabrero D, Company C, Urquiza JM, Tegnér J, Rodríguez-Gallego C, López-Granados E, Ballestar E - Nat Commun (2015)

Validation of DNA methylation results by bisulfite pyrosequencing.(a) Schemes depicting a selection of genes indicating the differentially methylated CpG site (red line) and its relative location with respect to the TSS (located with an arrow). Additional CpG sites are represented with vertical black lines. CpG islands are represented with a green bar. (b) Bisulfite pyrosequencing of selected genes, all of which are relevant to B-cell biology. In addition to CD19+ B lymphocytes, CD4+ T lymphocytes and CD14+ cells (monocytes) were analysed for all genes. Methylation levels are represented as percentages. Bisulfite sequencing was technical triplicates of each sibling of the MWZ twin pair. Error bars correspond to s.d. Student's t-test comparisons with a P value above 0.05 are considered with a nonsignificant difference, and are not presented (c) β-values obtained from the DNA methylation array. The B cells from CVID twin have higher levels of DNA methylation (Δβ≥0.1) in the analysed genes than those from his healthy sibling. Black filled circles and black filled triangles, respectively, indicate the three β-values for the control twin and the CVID twin. The black line indicates the average of the β-values in each condition.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4557293&req=5

f2: Validation of DNA methylation results by bisulfite pyrosequencing.(a) Schemes depicting a selection of genes indicating the differentially methylated CpG site (red line) and its relative location with respect to the TSS (located with an arrow). Additional CpG sites are represented with vertical black lines. CpG islands are represented with a green bar. (b) Bisulfite pyrosequencing of selected genes, all of which are relevant to B-cell biology. In addition to CD19+ B lymphocytes, CD4+ T lymphocytes and CD14+ cells (monocytes) were analysed for all genes. Methylation levels are represented as percentages. Bisulfite sequencing was technical triplicates of each sibling of the MWZ twin pair. Error bars correspond to s.d. Student's t-test comparisons with a P value above 0.05 are considered with a nonsignificant difference, and are not presented (c) β-values obtained from the DNA methylation array. The B cells from CVID twin have higher levels of DNA methylation (Δβ≥0.1) in the analysed genes than those from his healthy sibling. Black filled circles and black filled triangles, respectively, indicate the three β-values for the control twin and the CVID twin. The black line indicates the average of the β-values in each condition.
Mentions: To confirm the results obtained from the DNA methylation arrays, we performed bisulfite pyrosequencing of a selection of the aforementioned genes (Table 1), hypermethylated in B cells isolated from the same pair of twins discordant for CVID. In brief, the selection of genes was carried out among those displaying the largest changes in DNA methylation between the CVID and healthy sibling, and also to cover different functions in B-cell biology: PIK3CD, RPS6KB2 encode proteins involved in the cell proliferation, cell growth and protein synthesis, that occur in response to BCR signalling; BCL2L1 encodes a protein with anti-apoptotic effects. TCF3 encodes a transcription factor essential in the establishment of B-cell identity. PTPRCAP encodes a protein necessary for proper phosphatase activity of CD45 during BCR signalling, and CORO1B, KCNN4 and KCNC4 encode proteins that are involved in Ca+2 signalling. We focused on looking at the same CpG sites for which significant changes had been identified (Fig. 2a). Despite the modest differences in DNA methylation measured by the initial high-throughput strategy, we confirmed by bisulfite pyrosequencing a robust increase in DNA methylation in the CVID sibling with respect to the healthy sibling (Fig. 2b), with very similar values to those obtained using bead arrays (Fig. 2c). We also tested the DNA methylation changes in CD4+ cells (T lymphocytes) and CD14+ cells (monocytes) from the same individuals (Fig. 2b). In contrast with the robust increase in the frequency of B cells from the CVID sibling with respect to the healthy sibling, we observed no differences or changes in the opposite direction in T cells and monocytes, highlighting the specificity of the observed CVID-associated hypermethylation of these genes to B cells.

Bottom Line: Common variable immunodeficiency (CVID), the most frequent primary immunodeficiency characterized by loss of B-cell function, depends partly on genetic defects, and epigenetic changes are thought to contribute to its aetiology.Individual analysis confirms hypermethylation of these genes.Our results not only indicate a role for epigenetic alterations in CVID but also identify relevant DNA methylation changes in B cells that could explain the clinical manifestations of CVID individuals.

View Article: PubMed Central - PubMed

Affiliation: Chromatin and Disease Group, Cancer Epigenetics and Biology Programme (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Avda Gran Via 199-203, 08908L'Hospitalet de Llobregat, Barcelona, Spain.

ABSTRACT
Common variable immunodeficiency (CVID), the most frequent primary immunodeficiency characterized by loss of B-cell function, depends partly on genetic defects, and epigenetic changes are thought to contribute to its aetiology. Here we perform a high-throughput DNA methylation analysis of this disorder using a pair of CVID-discordant MZ twins and show predominant gain of DNA methylation in CVID B cells with respect to those from the healthy sibling in critical B lymphocyte genes, such as PIK3CD, BCL2L1, RPS6KB2, TCF3 and KCNN4. Individual analysis confirms hypermethylation of these genes. Analysis in naive, unswitched and switched memory B cells in a CVID patient cohort shows impaired ability to demethylate and upregulate these genes in transitioning from naive to memory cells in CVID. Our results not only indicate a role for epigenetic alterations in CVID but also identify relevant DNA methylation changes in B cells that could explain the clinical manifestations of CVID individuals.

No MeSH data available.


Related in: MedlinePlus