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Combined immunodeficiency develops with age in Immunodeficiency-centromeric instability-facial anomalies syndrome 2 (ICF2).

von Bernuth H, Ravindran E, Du H, Fröhler S, Strehl K, Krämer N, Issa-Jahns L, Amulic B, Ninnemann O, Xiao MS, Eirich K, Kölsch U, Hauptmann K, John R, Schindler D, Wahn V, Chen W, Kaindl AM - Orphanet J Rare Dis (2014)

Bottom Line: ICF2, caused by biallelic ZBTB24 gene mutations, is acknowledged primarily as an isolated B-cell defect.Here, we extend the phenotype spectrum by describing, in particular, for the first time the development of a combined immune defect throughout the disease course as well as putative autoimmune phenomena such as granulomatous hepatitis and nephritis.We also demonstrate impaired cell-proliferation and increased cell death of immune and non-immune cells as well as data suggesting a chromosome separation defect in addition to the known chromosome condensation defect.

View Article: PubMed Central - PubMed

Affiliation: Pediatric Pneumology and Immunology, Charité - Universitätsmedizin Berlin, Augustenburger Platz 1, 13353, Berlin, Germany. horst.von-bernuth@charite.de.

ABSTRACT
The autosomal recessive immunodeficiency-centromeric instability-facial anomalies syndrome (ICF) is characterized by immunodeficiency, developmental delay, and facial anomalies. ICF2, caused by biallelic ZBTB24 gene mutations, is acknowledged primarily as an isolated B-cell defect. Here, we extend the phenotype spectrum by describing, in particular, for the first time the development of a combined immune defect throughout the disease course as well as putative autoimmune phenomena such as granulomatous hepatitis and nephritis. We also demonstrate impaired cell-proliferation and increased cell death of immune and non-immune cells as well as data suggesting a chromosome separation defect in addition to the known chromosome condensation defect.

No MeSH data available.


Related in: MedlinePlus

Cellular defects in patient fibroblasts and lymphoblastoid cells, reproduced in HEK cells through siRNA and expression of mutant ZBTB24. (A) Reduced cell viability (n = 8 per time period, Student’s t-test), (B) reduced proliferation (n = 8, 36 h after plating, Student’s t-test), and (C) increased apoptosis of ZBTB24 mutant fibroblasts (activated caspase 3/7 per cell viability; n = 8, Student’s t-test). (D) Abnormal spindle (α-tubulin) formation with increase of slightly broader, unfocused microtubules poles (n = 100 metaphase LCLs, Student’s t-test, view Additional file 9: Figures S3 for further images throughout the cell cycle and specifically in metaphase cells) and (E, F) strongly reduced fluorescence signals of centrosomal marker CDK5RAP2 (but not γ-tubulin) in patient lyphoblastoid cells (n = 105 metaphase cells, Student’s t-test); total γ-tubulin levels were unaltered (n = 3, Student’s t-test). Scale bar 5 μm. View Additional file 10: Figures S4 for further images throughout the cell cycle and specifically in metaphase cells. (G) Reduced cell viability (n = 8 per time period, One-way ANOVA) and (H) mitotic spindles defect in HEK cells expressing mutant (c.1222 T > G) ZBTB24. Abnormal spindle formation with slightly broader, unfocused microtubule poles in mutant cells. Scale bar 5 μm. View Additional file 11: Figure S5 for further images throughout the cell cycle, qPCR and Western blot results. (I)ZBTB24 mRNA downregulation in HEK through siRNA (qPCR, Student’s t-test) causes (J) reduced cell culture growth (n = 3 per group per time period; Student’s t-test). ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001.
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Fig2: Cellular defects in patient fibroblasts and lymphoblastoid cells, reproduced in HEK cells through siRNA and expression of mutant ZBTB24. (A) Reduced cell viability (n = 8 per time period, Student’s t-test), (B) reduced proliferation (n = 8, 36 h after plating, Student’s t-test), and (C) increased apoptosis of ZBTB24 mutant fibroblasts (activated caspase 3/7 per cell viability; n = 8, Student’s t-test). (D) Abnormal spindle (α-tubulin) formation with increase of slightly broader, unfocused microtubules poles (n = 100 metaphase LCLs, Student’s t-test, view Additional file 9: Figures S3 for further images throughout the cell cycle and specifically in metaphase cells) and (E, F) strongly reduced fluorescence signals of centrosomal marker CDK5RAP2 (but not γ-tubulin) in patient lyphoblastoid cells (n = 105 metaphase cells, Student’s t-test); total γ-tubulin levels were unaltered (n = 3, Student’s t-test). Scale bar 5 μm. View Additional file 10: Figures S4 for further images throughout the cell cycle and specifically in metaphase cells. (G) Reduced cell viability (n = 8 per time period, One-way ANOVA) and (H) mitotic spindles defect in HEK cells expressing mutant (c.1222 T > G) ZBTB24. Abnormal spindle formation with slightly broader, unfocused microtubule poles in mutant cells. Scale bar 5 μm. View Additional file 11: Figure S5 for further images throughout the cell cycle, qPCR and Western blot results. (I)ZBTB24 mRNA downregulation in HEK through siRNA (qPCR, Student’s t-test) causes (J) reduced cell culture growth (n = 3 per group per time period; Student’s t-test). ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001.

Mentions: By whole exome sequencing, we identified the homozygous missense mutation c.1222 T > G of the ZBTB24 gene (NM_014797) in the index patient inherited from the healthy parents (Figure 1C). This previously described mutation alters evolutionarily conserved amino acids in a highly conserved zinc finger domain (p.C408G; Figure 1D,E, Additional file 1: Table S1). Patients with the c.1222 T > G mutation show a variable phenotype, arguing against a clear genotype-phenotype correlation and for a residual activity of mutant ZBTB24. In line with this, ZBTB24 mRNA levels did not differ significantly between patient and control (Additional file 8: Figure S2). Chromosome metaphase preparations revealed increased rates of undercondensated juxta-centromeric chromosomes 1q, 16q, and less frequent of 9q regions, characteristic for ICF2 (Figure 1F). This further increased upon exposure of cultures to 5-azacytidine (DNA methylation interfering agent), eventually resulting in chromosome instability (data not shown). Because ZBTB24 not only impacts on immune cells, we examined patient fibroblasts and detected significantly reduced proliferation, increased apoptosis and discrete spindle defects (broader and unfocused microtubule poles; Figure 2A-D, Additional file 9: Figure S3). In mutant cells, centrosomal CDK5RAP2 was strongly reduced, while centrosomal y-tubulin staining and total y-tubulin levels were normal (Figure 2E,F, Additional file 10: Figure S4). The mechanisms underlying the reduction in CDK5RAP2, which is associated with stem cell proliferation and microcephaly with intellectual deficit [14], may be involved in the pathogenesis of the neurological phenotype of ICF.Figure 2


Combined immunodeficiency develops with age in Immunodeficiency-centromeric instability-facial anomalies syndrome 2 (ICF2).

von Bernuth H, Ravindran E, Du H, Fröhler S, Strehl K, Krämer N, Issa-Jahns L, Amulic B, Ninnemann O, Xiao MS, Eirich K, Kölsch U, Hauptmann K, John R, Schindler D, Wahn V, Chen W, Kaindl AM - Orphanet J Rare Dis (2014)

Cellular defects in patient fibroblasts and lymphoblastoid cells, reproduced in HEK cells through siRNA and expression of mutant ZBTB24. (A) Reduced cell viability (n = 8 per time period, Student’s t-test), (B) reduced proliferation (n = 8, 36 h after plating, Student’s t-test), and (C) increased apoptosis of ZBTB24 mutant fibroblasts (activated caspase 3/7 per cell viability; n = 8, Student’s t-test). (D) Abnormal spindle (α-tubulin) formation with increase of slightly broader, unfocused microtubules poles (n = 100 metaphase LCLs, Student’s t-test, view Additional file 9: Figures S3 for further images throughout the cell cycle and specifically in metaphase cells) and (E, F) strongly reduced fluorescence signals of centrosomal marker CDK5RAP2 (but not γ-tubulin) in patient lyphoblastoid cells (n = 105 metaphase cells, Student’s t-test); total γ-tubulin levels were unaltered (n = 3, Student’s t-test). Scale bar 5 μm. View Additional file 10: Figures S4 for further images throughout the cell cycle and specifically in metaphase cells. (G) Reduced cell viability (n = 8 per time period, One-way ANOVA) and (H) mitotic spindles defect in HEK cells expressing mutant (c.1222 T > G) ZBTB24. Abnormal spindle formation with slightly broader, unfocused microtubule poles in mutant cells. Scale bar 5 μm. View Additional file 11: Figure S5 for further images throughout the cell cycle, qPCR and Western blot results. (I)ZBTB24 mRNA downregulation in HEK through siRNA (qPCR, Student’s t-test) causes (J) reduced cell culture growth (n = 3 per group per time period; Student’s t-test). ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001.
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Fig2: Cellular defects in patient fibroblasts and lymphoblastoid cells, reproduced in HEK cells through siRNA and expression of mutant ZBTB24. (A) Reduced cell viability (n = 8 per time period, Student’s t-test), (B) reduced proliferation (n = 8, 36 h after plating, Student’s t-test), and (C) increased apoptosis of ZBTB24 mutant fibroblasts (activated caspase 3/7 per cell viability; n = 8, Student’s t-test). (D) Abnormal spindle (α-tubulin) formation with increase of slightly broader, unfocused microtubules poles (n = 100 metaphase LCLs, Student’s t-test, view Additional file 9: Figures S3 for further images throughout the cell cycle and specifically in metaphase cells) and (E, F) strongly reduced fluorescence signals of centrosomal marker CDK5RAP2 (but not γ-tubulin) in patient lyphoblastoid cells (n = 105 metaphase cells, Student’s t-test); total γ-tubulin levels were unaltered (n = 3, Student’s t-test). Scale bar 5 μm. View Additional file 10: Figures S4 for further images throughout the cell cycle and specifically in metaphase cells. (G) Reduced cell viability (n = 8 per time period, One-way ANOVA) and (H) mitotic spindles defect in HEK cells expressing mutant (c.1222 T > G) ZBTB24. Abnormal spindle formation with slightly broader, unfocused microtubule poles in mutant cells. Scale bar 5 μm. View Additional file 11: Figure S5 for further images throughout the cell cycle, qPCR and Western blot results. (I)ZBTB24 mRNA downregulation in HEK through siRNA (qPCR, Student’s t-test) causes (J) reduced cell culture growth (n = 3 per group per time period; Student’s t-test). ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001.
Mentions: By whole exome sequencing, we identified the homozygous missense mutation c.1222 T > G of the ZBTB24 gene (NM_014797) in the index patient inherited from the healthy parents (Figure 1C). This previously described mutation alters evolutionarily conserved amino acids in a highly conserved zinc finger domain (p.C408G; Figure 1D,E, Additional file 1: Table S1). Patients with the c.1222 T > G mutation show a variable phenotype, arguing against a clear genotype-phenotype correlation and for a residual activity of mutant ZBTB24. In line with this, ZBTB24 mRNA levels did not differ significantly between patient and control (Additional file 8: Figure S2). Chromosome metaphase preparations revealed increased rates of undercondensated juxta-centromeric chromosomes 1q, 16q, and less frequent of 9q regions, characteristic for ICF2 (Figure 1F). This further increased upon exposure of cultures to 5-azacytidine (DNA methylation interfering agent), eventually resulting in chromosome instability (data not shown). Because ZBTB24 not only impacts on immune cells, we examined patient fibroblasts and detected significantly reduced proliferation, increased apoptosis and discrete spindle defects (broader and unfocused microtubule poles; Figure 2A-D, Additional file 9: Figure S3). In mutant cells, centrosomal CDK5RAP2 was strongly reduced, while centrosomal y-tubulin staining and total y-tubulin levels were normal (Figure 2E,F, Additional file 10: Figure S4). The mechanisms underlying the reduction in CDK5RAP2, which is associated with stem cell proliferation and microcephaly with intellectual deficit [14], may be involved in the pathogenesis of the neurological phenotype of ICF.Figure 2

Bottom Line: ICF2, caused by biallelic ZBTB24 gene mutations, is acknowledged primarily as an isolated B-cell defect.Here, we extend the phenotype spectrum by describing, in particular, for the first time the development of a combined immune defect throughout the disease course as well as putative autoimmune phenomena such as granulomatous hepatitis and nephritis.We also demonstrate impaired cell-proliferation and increased cell death of immune and non-immune cells as well as data suggesting a chromosome separation defect in addition to the known chromosome condensation defect.

View Article: PubMed Central - PubMed

Affiliation: Pediatric Pneumology and Immunology, Charité - Universitätsmedizin Berlin, Augustenburger Platz 1, 13353, Berlin, Germany. horst.von-bernuth@charite.de.

ABSTRACT
The autosomal recessive immunodeficiency-centromeric instability-facial anomalies syndrome (ICF) is characterized by immunodeficiency, developmental delay, and facial anomalies. ICF2, caused by biallelic ZBTB24 gene mutations, is acknowledged primarily as an isolated B-cell defect. Here, we extend the phenotype spectrum by describing, in particular, for the first time the development of a combined immune defect throughout the disease course as well as putative autoimmune phenomena such as granulomatous hepatitis and nephritis. We also demonstrate impaired cell-proliferation and increased cell death of immune and non-immune cells as well as data suggesting a chromosome separation defect in addition to the known chromosome condensation defect.

No MeSH data available.


Related in: MedlinePlus