Mutations in ZBTB24 are associated with immunodeficiency, centromeric instability, and facial anomalies syndrome type 2

Abstract

Autosomal-recessive immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is mainly characterized by recurrent, often fatal, respiratory and gastrointestinal infections. About 50% of patients carry mutations in the DNA methyltransferase 3B gene (DNMT3B) (ICF1). The remaining patients carry unknown genetic defects (ICF2) but share with ICF1 patients the same immunological and epigenetic features, including hypomethylation of juxtacentromeric repeat sequences. We performed homozygosity mapping in five unrelated ICF2 patients with consanguineous parents and then performed whole-exome sequencing in one of these patients and Sanger sequencing in all to identify mutations in the zinc-finger- and BTB (bric-a-bric, tramtrack, broad complex)-domain-containing 24 (ZBTB24) gene in four consanguineously descended ICF2 patients. Additionally, we found ZBTB24 mutations in an affected sibling pair and in one patient for whom it was not known whether his parents were consanguineous. ZBTB24 belongs to a large family of transcriptional repressors that include members, such as BCL6 and PATZ1, with prominent regulatory roles in hematopoietic development and malignancy. These data thus indicate that ZBTB24 is involved in DNA methylation of juxtacentromeric DNA and in B cell development and/or B and T cell interactions. Because ZBTB24 is a putative DNA-binding protein highly expressed in the lymphoid lineage, we predict that by studying the molecular function of ZBTB24, we will improve our understanding of the molecular pathophysiology of ICF syndrome and of lymphocyte biology in general.

Figure 1

Genetics and Epigenetics of ICF2 Patients (A) Pedigrees of 11 ICF2 families studied. The parents of P1, P2, P3, and P4 were first-degree relatives. The great-grandmothers of patient P5 were sisters. The history of consanguinity for P10 was unknown before this study. However, our SNP array data show large regions of homozygosity, suggesting that the parents of P10 are related (data not shown). (B) α-satellite hypomethylation in ICF2 patients. Southern-blot analysis of the α-satellite repeat on chromosome 9 in a control individual, an ICF1 patient with a DNMT3B mutation, two ICF2 patients (P1 and P5) with a mutation in ZBTB24, and one ICF2 patient (P4) in whom no mutations in DNMT3B or ZBTB24 were identified and in whom the DNMT3B and ZBTB24 loci were excluded by homozygosity mapping. DNA samples (2 μg) were digested overnight with the restriction enzyme HhaI, separated by linear gel electrophoresis on a 0.8% agarose gel. Subsequently, Southern blotting of the DNA and hybridization of the resulting membrane with a probe targeted to the α-satellite repeat on chromosome 9 were performed.

Figure 2

ZBTB24 Mutations at the DNA and Protein Level (A) Sanger sequencing results of seven ICF2 patients with ZBTB24 mutations and an unrelated control individual. P1 carries a homozygous 1 bp deletion resulting in a frameshift and a premature stop codon three amino acids downstream (p.Asn306IlefsX4). Both parents are heterozygous carriers for the mutation, and the healthy brother is heterozygous as well (data not shown). P2 is homozygous for a nonsense mutation resulting in a serine-to-stop conversion (p.Ser16X). P3 carries a homozygous sequence variant creating a premature stop codon (p.Arg320X). Both parents are heterozygous carriers for the mutation (data not shown). P5 carries a homozygous 1 bp insertion resulting in a frameshift and a premature stop codon 27 amino acids downstream (p.Val168SerfsX28). P6 and P7 are compound heterozygous for a serine-to-stop nonsense mutation (p.Ser278X) and a cysteine-to-glycine missense mutation (p.Cys408Gly). P10 is homozygous for a nonsense mutation resulting in an arginine-to-stop conversion (p.Arg457X). The mother is a heterozygous carrier for the mutation (data not shown). (B) A schematic of the two isoforms of the ZBTB24 protein. The full-length ZBTB24 protein (isoform 1) contains a BTB domain (amino acids 10–133), a DNA-binding A-T hook domain (amino acids 159–171), and eight C₂H₂ zinc finger domains of 23 amino acids each (amino acids 294–512). The shorter isoform 2 lacks seven C₂H₂ zinc finger domains, and the last 15 amino acids are an alternative sequence (an alternative stop codon is used in the intron between exon 2 and exon 3). RNA ZBTB24 denotes the location of the primers used for RT-PCR. At least eight SNPs have been found in the coding sequence of ZBTB24. The numbers and locations of these SNPs are indicated as well.

Figure 3

ZBTB24 Expression Full-length ZBTB24 expression levels were measured in human primary cell cultures (ES cells and fibroblasts; n = 2), in human tissues (alveoli, kidney, liver, and thymus; n = 1), in human cell lines (HeLa, HCT116, JVM-2, Jurkat, LCL, MOLT-4, Raji, Ramos, Tera-2, and THP-1; n = 1), in human B cells (naive B cells [CD19⁺CD27⁻IgD⁺)], unswitched memory B cells [CD19⁺CD27⁺IgD⁺IgM⁺], switched memory B cells [CD19⁺CD27⁺IgD⁻IgM⁻], and total B cells [CD19⁺]; n = 2), in human CD4⁺ T cells (naive CD4⁺ T cells [CD4⁺CD45RA⁺CCR7⁺], memory CD4⁺ T cells [CD4⁺CD45RA⁻], and total CD4⁺ T cells [CD4⁺]; n = 2), and in human NK cells (CD3⁻CD56⁺; n = 2). Expression levels were measured in duplicate and are presented relative to the expression levels of the three housekeeping genes GAPDH, GUSB, and ACTB, which were also measured in duplicate. Data are presented as mean ± standard deviation.