ICF, an immunodeficiency syndrome: DNA methyltransferase 3B involvement, chromosome anomalies, and gene dysregulation

Figures & data

Figure 1 Hypomethylated DNA in constitutive heterochromatin in ICF. Cartoon illustrating the constitutive heterochromatin regions that display ICF-specific hypomethylation and chromosome abnormalities. Dark gray box, juxtacentromeric (pericentromeric) heterochromatin; white box, centromere.

Table I.  Immune system-related genes with significant differences in RNA levels in ICF vs. control lymphoblasts.

				Microarray data^a					qRT-PCR data^b
Abbreviated gene name	Ref seq. ID or other ID	Chrom. location	Description	Array expt. no.	Mean for ICF	Mean for controls	FC	P-value from t-test	FC	P-value from t-test
IGHG3	M87789	14q32.33	Immunoglobulin heavy constant gamma 3	1	291	>18,600	− 60	0.004	NA
	211647_x_at		Ig rearranged mu-chain gene V-N-D-N-J-region, complete cds	2	62	8765	− 100	2 × 10^− 8
NT5E (CD73)	NM_002526	6q14-q21	5′-Nucleotidase, ecto; hydrolyzes extracellular nucleotides; intracellular signalling, lymphocyte proliferation, activation, and cell cohesion; much greater activity in mature than immature B and T cells	2	377	158	+2.4	0.005	NA
MAP4K4	NM_145687	2q11.2	Mitogen-activated protein kinase kinase kinase kinase 4; role in T-cell antigen-mediated responses, in cell motility, and in TNF-α/JNK and p53 signalling pathways	2	890	14	+18	0.001	NA
CASP1	NM_033292	11q22.3	Caspase 1, apoptosis-related cysteine peptidase (interleukin 1, beta, convertase); in B cells, activation of NF-κB and MAPK14	2	961	333	+3.0	0.009	NA
CD27	NM_001242	12p13	Tumor necrosis factor receptor; family, member 7 (TNFRSF7); T-cell and B-cell proliferation response to antigens; somatically mutated, germinal center, memory, and plasma cells	1	1439	2890	− 2.0	0.04	NA
				2	1552	3081	− 2.0	0.03
ITGB2 (CD18)	NM_000211	21q22.3	Integrin, beta 2 (complement component 3 receptor 3 and 4 subunit); cell surface adhesion receptor involved in B-cell differentiation and homing	2	1046	357	+2.9	0.03	NA
CD44	NM_000610	11p13	CD44 antigen; attachment to endothelial cells and homing to peripheral lymphoid organs and sites of inflammation; naïve and memory B cells, not on germinal center B cells	2	4651	1438	+3.2	0.005	+3.5	0.01
CD40	NM_001250	20q12-q13.2	Tumor necrosis factor receptor superfamily, member 5; interaction with its T-cell ligand needed for B-cell proliferation, differentiation, isotype switching, humoral memory response; mature B cells	2	1460	487	+3.0	0.01	NA
CKLF	NM_016326	16q22.1	Chemokine-like factor; chemotaxis; upregulated in activated T cells but not in B cells; very high expression in testes	2	1599	351	+4.6	0.003	+3.3	0.03

^a The first [5] and second experiments on ICF vs.control B-cell lines were done similarly on HuGeneFL and HG-U133 microarrays, respectively (Affymetrix). The ICF LCLs for Expt. #2 were those in Expt. #1 (only one of the two ICF B stocks) with three additional LCLs, patients 1 and 3 from one study [29] and patient 5 from another [6]. The control LCLs for Expt. #2 were from the mothers and fathers of patient ICF B and C and AG15022 and AG14953 (Coriell Institute). A positive fold change (FC) denotes RNA overexpression in ICF and is the mean signal for ICF divided by that for the controls for that probe set. A negative FC indicates underexpression in ICF and is the negative of the mean signal for controls divided by that for ICF. P-values are for two-sample t-tests to evaluate the significance of ICF-associated increases or decreases in mean RNA levels relative to the controls. For IGHG3, PRKCH, CD44, and CKLF, another probe set in the microarray gave similar results in Expt. #2 (not shown)

^b Real-time quantitative RT-PCR (MyIQ Cycler and iQ SYBR Green Supermix, Bio-Rad) was done on all the ICF LCL samples used for Expt. #2 and 6–12 control LCLs (including additional B-cell lines) that were prepared from random hexanucleotide or oligo(dT)-primed cDNA. Primers were designed for the HG-U133 microarray probe regions and optimal annealing temperatures for PCR were determined by gradient PCR. The data were normalized to those from GAPDH. The fold change is described as for the microarray data. The P-values are for the differences in the mean RNA levels using log-transformed data. NA, not assayed.

Table II.  More genes with significantly altered RNA levels in ICF vs. control lymphoblasts.

				Microarray data^a					qRT-PCR data^a
Gene symbol	Ref seq. ID	Chrom. location	Description	Array expt. no.	Mean for ICF	Mean for controls.	FC	P-value from t-test	FC	P-value from t-test
PRKCH	NM_006255	14q22-q23	Protein kinase C, eta; calcium-independent and phospholipid-dependent; a PRKCH cleavage product is implicated in apoptosis in pro-B cells but expression in T cells is much higher than in B cells; expression correlated with NO production	1	379	100	+3.8	0.04
				2	2301	170	+13.6	0.003	+6.5	0.04
GUCY1A3	NM_000856	4q32.1	Guanylate cyclase 1, soluble, alpha 3; heterodimerizes with beta subunit to give an enzyme (sGC) strongly activated by NO	1	233	101	+2.3	0.03
				2	1193	417	+2.9	7 × 10^− 5	+6.8	4 × 10^− 6
GUCY1B3	NM_000857	4q32.1	Guanylate cyclase 1, soluble, beta 3; heme-containing subunit of sGC; MAPK13 is a downstream effector and HMOX1 is upstream	1	1316	292	+4.5	0.007
				2	3759	782	+4.8	0.001	+10.6	1 × 10^− 5
HMOX1	NM_002133	22q13.1	Heme oxygenase (decycling) 1; ubiquitious transcriptionally inducible stress protein; degrades heme; generates CO for anti-proliferative effects on T cells	1	577	2353	− 4.1	0.03
				2	864	1559	− 1.8	0.009	NA
MAPK13	NM_002754	6p21.31	Mitogen-activated protein kinase 13; stress activated kinase; activated by PRKCH during regulation of keratinocyte differentiation; downstream in the sGC pathway; is pro-apoptotic; implicated in antigen-meidated T-cell responses	2	388	104	+3.7	0.006	NA
PTPN13	NM_080683	4q21.3	Protein tyrosine phosphatase, non-receptor type 13 (APO-1/CD95 (Fas)-associated phosphatase); both anti- and pro-apoptic effects; I κBα is a substrate; high expression in placenta and testis	1	271	41	+2.7	0.02
				2	623	53	+12	1 × 10^− 7	+5.0	0.02
BCL2L10	NM_020396	15q21.2	BCL2-like 10 (apoptosis facilitator); both anti- and pro-apototic	2	560	184	+3.0	0.002	NA
CNN3	NM_001839	1p22-p21	Calponin 3, acidic; actin-binding protein; low expression in leukocytes	1	407	80	+4.1	0.008	NA
				2	1046	357	+2.9	0.03
SLC1A1	NM_004170	9p24	Solute carrier family 1 (neuronal/epithelial high affinity glutamate transporter)	1	137	237	− 1.7	0.001	NA
				2	716	1493	− 2.1	0.007	NA
NR2F2	NM_021005	15q26.2	Nuclear receptor subfamily 2, group F, member 2; COUP-TFII; negative post transcriptional regulator of MYOD1	2	824	91	+9.0	0.003	+2.9	0.005
SMARCA2	NM_003070	9p24.3	SWI/SNF related, matrix associated, subfamily a, member 2; BRM; chromatin remodelling	2	2814	1250	+2.1	0.002	+3.3	0.004

^a See the footnotes to Table I. Two probe sets each for GUCY1B3, CNN3, SMARCA2, and BCL2L10 in microarray Expt. #2 gave similar results.

Figure 2 Analysis of DNA methylation upstream of PTPN13. Representative COBRA analysis for DNA methylation of a gene that had RNA upregulated in ICF vs. control LCLs. DNA samples had been modified with bisulfite and amplified by PCR with primers at the indicated positions as previously described [5]. The PCR products could be cleaved by TaqI or BstUI only if they had been methylated at the CpG dinucleotide in the indicated sites in genomic DNA; the pre-TaqI site, CCGA, would be converted to a TaqI site, TCGA, upon bisulfite treatment and PCR if it was Cm5 CGA in genomic DNA. (A) Diagram of the 5′PTPN region showing the transcription start site (TSS) [123] at Chr4 87,734,909 (hg18, UCSC database), the 5′CpG island ( − 701 to − 150), and PCR primers; positions are given relative to the TSS. (B) and (C), electrophoresis gels stained with ethidium bromide and visualized for fluorescent bands from the PCR products ( − 628 to − 119 and − 1250 to − 940) digested with BstUI or TaqI. PBMC, peripheral blood mononuclear cells; ICF LCLs are described in the legend to Table I, with the addition of another control LCL (AG14832, Coriell Institute). Sizes are given in bp for the expected and obtained restriction products.

Ehrlich M, Buchanan K, Tsien F, Jiang G, Sun B, Uicker W, Weemaes C, Smeets D, Sperling K, Belohradsky B, et al. DNA methyltransferase 3B mutations linked to the ICF syndrome cause dysregulation of lymphogenesis genes. Hum Mol Gen 2001; 10: 2917–2931

 PubMed Web of Science ®Google Scholar

Hernandez R, Frady A, Zhang X-Y, Varela M, Ehrlich M. Preferential induction of chromosome 1 multibranched figures and whole-arm deletions in a human pro-B cell line treated with 5-azacytidine or 5-azadeoxycytidine. Cytogenet Cell Genet 1997; 76: 196–201

 Google Scholar

Wijmenga C, Hansen RS, Gimelli G, Bjorck EJ, Davies EG, Valentine D, Belohradsky BH, van Dongen JJ, Smeets DF, van den Heuvel LP, et al. Genetic variation in ICF syndrome: Evidence for genetic heterogeneity. Hum Mutat 2000; 16: 509–517

 PubMed Web of Science ®Google Scholar

Saras J, Claesson-Welsh L, Heldin CH, Gonez LJ. Cloning and characterization of PTPL1, a protein tyrosine phosphatase with similarities to cytoskeletal-associated proteins. J Biol Chem 1994; 269: 24082–24089

 PubMed Web of Science ®Google Scholar