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

Abstract

The immunodeficiency, centromeric region instability, and facial anomalies syndrome (ICF) is the only disease known to result from a mutated DNA methyltransferase gene, namely, DNMT3B. Characteristic of this recessive disease are decreases in serum immunoglobulins despite the presence of B cells and, in the juxtacentromeric heterochromatin of chromosomes 1 and 16, chromatin decondensation, distinctive rearrangements, and satellite DNA hypomethylation. Although DNMT3B is involved in specific associations with histone deacetylases, HP1, other DNMTs, chromatin remodelling proteins, condensin, and other nuclear proteins, it is probably the partial loss of catalytic activity that is responsible for the disease. In microarray experiments and real-time RT-PCR assays, we observed significant differences in RNA levels from ICF vs. control lymphoblasts for pro- and anti-apoptotic genes (BCL2L10, CASP1, and PTPN13); nitrous oxide, carbon monoxide, NF-kappaB, and TNFalpha signalling pathway genes (PRKCH, GUCY1A3, GUCY1B3, MAPK13; HMOX1, and MAP4K4); and transcription control genes (NR2F2 and SMARCA2). This gene dysregulation could contribute to the immunodeficiency and other symptoms of ICF and might result from the limited losses of DNA methylation although ICF-related promoter hypomethylation was not observed for six of the above examined genes. We propose that hypomethylation of satellite 2 at 1qh and 16qh might provoke this dysregulation gene expression by trans effects from altered sequestration of transcription factors, changes in nuclear architecture, or expression of noncoding RNAs.

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.

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 aTaqI 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 Bst UI or Taq I. 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.