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Background: Loss-of-function mutations of ZBTB24 cause immunodeficiency, centromeric instability, and facial anomalies syndrome 2 (ICF2). ICF2 is a rare autosomal recessive disorder with immunological defects in serum antibodies and circulating memory B cells, resulting in recurrent and sometimes fatal respiratory and gastrointestinal infections. The genotype–phenotype correlation in patients with ICF2 indicates an essential role of ZBTB24 in the terminal differentiation of B cells. Methods: We used the clustered regularly interspaced short palindromic repeats (CRISPER)/Cas9 technology to generate B cell specific Zbtb24-deficient mice and verified the deletion specificity and efficiency by quantitative polymerase chain reaction (Q-PCR) and western blotting analyses in fluorescence-activated cell sorting (FACS)-sorted cells. The development, phenotype of B cells and in vivo responses to T cell dependent or independent antigens post immunization were analyzed by flow cytometry and enzyme-linked immunosorbent assay (ELISA). Adoptive transfer experiment in combination with in vitro cultures of FACS-purified B cells and RNA-Seq analysis were utilized to specifically determine the impact of Zbtb24 on B cell biology as well as the underlying mechanisms. Results: Zbtb24 is dispensable for B cell development and maintenance in naive mice. Surprisingly, B cell specific deletion of Zbtb24 does not evidently compromise germinal center reactions and the resulting primary and secondary antibody responses induced by T cell dependent antigens (TD-Ags), but significantly inhibits T cell independent antigen-elicited antibody productions in vivo. At the cellular level, Zbtb24-deficiency specifically impedes the plasma cell differentiation of B1 cells without impairing their survival, activation and proliferation in vitro. Mechanistically, Zbtb24-ablation attenuates heme biosynthesis partially through mTORC1 in B1 cells, and addition of exogenous hemin abrogates the differentiation defects of Zbtb24-null B1 cells. Conclusions: Zbtb24 seems to regulate antibody responses against TD-Ags B cell extrinsically, but it specifically promotes the plasma cell differentiation of B1 cells via heme synthesis in mice. Our study also suggests that defected B1 functions contribute to recurrent infections in patients with ICF2. [ABSTRACT FROM AUTHOR]

Abstract Background Loss-of-function mutations of ZBTB24 cause immunodeficiency, centromeric instability, and facial anomalies syndrome 2 (ICF2). ICF2 is a rare autosomal recessive disorder with immunological defects in serum antibodies and circulating memory B cells, resulting in recurrent and sometimes fatal respiratory and gastrointestinal infections. The genotype–phenotype correlation in patients with ICF2 indicates an essential role of ZBTB24 in the terminal differentiation of B cells. Methods We used the clustered regularly interspaced short palindromic repeats (CRISPER)/Cas9 technology to generate B cell specific Zbtb24-deficient mice and verified the deletion specificity and efficiency by quantitative polymerase chain reaction (Q-PCR) and western blotting analyses in fluorescence-activated cell sorting (FACS)-sorted cells. The development, phenotype of B cells and in vivo responses to T cell dependent or independent antigens post immunization were analyzed by flow cytometry and enzyme-linked immunosorbent assay (ELISA). Adoptive transfer experiment in combination with in vitro cultures of FACS-purified B cells and RNA-Seq analysis were utilized to specifically determine the impact of Zbtb24 on B cell biology as well as the underlying mechanisms. Results Zbtb24 is dispensable for B cell development and maintenance in naive mice. Surprisingly, B cell specific deletion of Zbtb24 does not evidently compromise germinal center reactions and the resulting primary and secondary antibody responses induced by T cell dependent antigens (TD-Ags), but significantly inhibits T cell independent antigen-elicited antibody productions in vivo. At the cellular level, Zbtb24-deficiency specifically impedes the plasma cell differentiation of B1 cells without impairing their survival, activation and proliferation in vitro. Mechanistically, Zbtb24-ablation attenuates heme biosynthesis partially through mTORC1 in B1 cells, and addition of exogenous hemin abrogates the differentiation defects of Zbtb24-null B1 cells. Conclusions Zbtb24 seems to regulate antibody responses against TD-Ags B cell extrinsically, but it specifically promotes the plasma cell differentiation of B1 cells via heme synthesis in mice. Our study also suggests that defected B1 functions contribute to recurrent infections in patients with ICF2.

Immunodeficiency, Centromeric instability and Facial anomalies (ICF) syndrome is a rare genetic disorder characterized by variable immunodeficiency. More than half of the affected individuals show mild to severe intellectual disability at early onset. This disorder is genetically heterogeneous and ZBTB24 is the causative gene of the subtype 2, accounting for about 30% of the ICF cases. ZBTB24 is a multifaceted transcription factor belonging to the Zinc-finger and BTB domain-containing protein family, which are key regulators of developmental processes. Aberrant DNA methylation is the main molecular hallmark of ICF syndrome. The functional link between ZBTB24 deficiency and DNA methylation errors is still elusive. Here, we generated a novel ICF2 disease model by deriving induced pluripotent stem cells (iPSCs) from peripheral CD34+-blood cells of a patient homozygous for the p.Cys408Gly mutation, the most frequent missense mutation in ICF2 patients and which is associated with a broad clinical spectrum. The mutation affects a conserved cysteine of the ZBTB24 zinc-finger domain, perturbing its function as transcriptional activator. ICF2-iPSCs recapitulate the methylation defects associated with ZBTB24 deficiency, including centromeric hypomethylation. We validated that the mutated ZBTB24 protein loses its ability to directly activate expression of CDCA7 and other target genes in the patient-derived iPSCs. Upon hematopoietic differentiation, ICF2-iPSCs showed decreased vitality and a lower percentage of CD34+/CD43+/CD45+ progenitors. Overall, the ICF2-iPSC model is highly relevant to explore the role of ZBTB24 in DNAmethylation homeostasis and provides a tool to investigate the early molecular events linking ZBTB24 deficiency to the ICF2 clinical phenotype. [ABSTRACT FROM AUTHOR]

Immunodeficiency, Centromeric instability and Facial anomalies (ICF) syndrome is a rare genetic disorder characterized by variable immunodeficiency. More than half of the affected individuals show mild to severe intellectual disability at early onset. This disorder is genetically heterogeneous and ZBTB24 is the causative gene of the subtype 2, accounting for about 30% of the ICF cases. ZBTB24 is a multifaceted transcription factor belonging to the Zinc-finger and BTB domain-containing protein family, which are key regulators of developmental processes. Aberrant DNA methylation is the main molecular hallmark of ICF syndrome. The functional link between ZBTB24 deficiency and DNA methylation errors is still elusive. Here, we generated a novel ICF2 disease model by deriving induced pluripotent stem cells (iPSCs) from peripheral CD34+-blood cells of a patient homozygous for the p.Cys408Gly mutation, the most frequent missense mutation in ICF2 patients and which is associated with a broad clinical spectrum. The mutation affects a conserved cysteine of the ZBTB24 zinc-finger domain, perturbing its function as transcriptional activator. ICF2-iPSCs recapitulate the methylation defects associated with ZBTB24 deficiency, including centromeric hypomethylation. We validated that the mutated ZBTB24 protein loses its ability to directly activate expression of CDCA7 and other target genes in the patient-derived iPSCs. Upon hematopoietic differentiation, ICF2-iPSCs showed decreased vitality and a lower percentage of CD34+/CD43+/CD45+ progenitors. Overall, the ICF2-iPSC model is highly relevant to explore the role of ZBTB24 in DNA methylation homeostasis and provides a tool to investigate the early molecular events linking ZBTB24 deficiency to the ICF2 clinical phenotype.

Since its discovery as a causative gene of the Immunodeficiency with Centromeric instability and Facial anomalies syndrome, ZBTB24 has emerged as a key player in DNA methylation, immunity and development. By extensively analyzing ZBTB24 genomic functions in ICF-relevant mouse and human cellular models, we document here its multiple facets as a transcription factor, with key roles in immune response-related genes expression and also in early embryonic development. Using a constitutive Zbtb24 ICF-like mutant and an auxin-inducible degron system in mouse embryonic stem cells, we showed that ZBTB24 is recruited to centromeric satellite DNA where it is required to establish and maintain the correct DNA methylation patterns through the recruitment of DNMT3B. The ability of ZBTB24 to occupy centromeric satellite DNA is conserved in human cells. Together, our results unveiled an essential and underappreciated role for ZBTB24 at mouse and human centromeric satellite repeat arrays by controlling their DNA methylation and transcription status., (© The Author(s) 2024. Published by Oxford University Press.)

Immunodeficiency, centromeric instability, facial anomalies (ICF) syndrome is a rare autosomal recessive disorder caused by mutations in either DNMT3B, ZBTB24, CDCA7, HELLS or an unknown gene(s). Among the known causative genes, ZBTB24 encodes a member of the BTB‐zinc finger (ZF) transcription factor family. The protein possesses a BTB domain, an AT‐hook and eight C2H2 ZF motifs. All ZBTB24 mutations reported in ICF patients are predicted to disrupt at least one ZF motif. Here, we show that both AT‐hook and distinct ZF motifs, particularly the 6th motif, of human and mouse ZBTB24 proteins are important for their heterochromatin localization. On the other hand, the 6th and 7th ZF motifs, and not the AT‐hook or the BTB domain, of the human and mouse proteins are essential for transcriptional activation of CDCA7, another ICF causative gene and a known target of ZBTB24. By deletion analysis of the human CDCA7 promoter, we show that two motifs for ZBTB24 binding are important for transcriptional activation of this gene. These results reveal the evolutionarily conserved domains and motifs important for the biological function of ZBTB24, which provides a basis for understanding the molecular mechanisms underlying the pathogenesis of ICF syndrome. [ABSTRACT FROM AUTHOR]

Increasing use of next‐generation sequencing technologies in clinical diagnostics allows large‐scale discovery of genetic variants, but also results in frequent identification of variants of unknown significance (VUSs). Their classification into disease‐causing and neutral variants is often hampered by the absence of robust functional tests. Here, we demonstrate that a luciferase reporter assay, in combination with ChIP‐qPCR, reliably separates pathogenic ZBTB24 missense variants in the context of immunodeficiency, centromeric instability, facial anomalies (ICF) syndrome from natural variants in healthy individuals and patients of other diseases. Application of our assay to two published ZBTB24 missense VUSs indicates that they are likely not to cause ICF2 syndrome. Furthermore, we show that rare gnomAD ZBTB24 missense variants in key residues of the C2H2‐ZF domain lead to a loss of function phenotype that resembles ICF2, suggesting that these individuals are carriers of ICF syndrome. In summary, we have developed a robust functional test to validate missense variants in ZBTB24. [ABSTRACT FROM AUTHOR]

Mutations in ZBTB24 and CDCA7 cause the Immunodeficiency, Centromeric Instability and Facial Anomalies syndrome type 2 and 3 (ICF2/3), respectively. Most ICF2 patients carry ZBTB24 nonsense mutations and are thus ZBTB24-deficient. Although the immune deficiency in ICF2 patients is primarily regarded as a B-cell defect due to the greatly reduced serum antibodies and circulating memory B cells, the reduced expansions of PBMCs stimulated by mitogens or recall antigens suggest a T-cell defect in these patients as well. However, the molecular mechanisms behind this T-cell dysfunction remain unknown. In the present study, we demonstrated that ZBTB24-deficiency significantly represses the proliferation of human T cells by promoting TRAIL-induced cell death. Downregulation of ZBTB24 in both Jurkat and human primary T cells upregulates the expression of TRAIL and/or its death receptors (TRAIL-R1/2), and induces significant amount of cells to undergo apoptosis. The profound survival defects of ZBTB24-deficient cells are largely reversed by blocking TRAIL/TRAIL-R interactions with exogenous recombinant TRAIL-R2. Moreover, ZBTB24-downregulation reduces the expression of CDCA7, and knockdown of the latter in human T cells results in a phenotype resembling that caused by ZBTB24-depletion. Functionally, overexpression of CDCA7 abrogates the increased apoptosis in ZBTB24-depleted Jurkat T cells. Together, these data indicated that ZBTB24 regulates human T-cell apoptosis via CDCA7/TRAIL-R axis. Our study thus not only provides a molecular explanation for the T-cell defects in ZBTB24-deficient ICF2 patients, but also highlights a convergence between ZBTB24 and CDCA7, the two ICF genes, in modulating the functions of T cells. • Knockdown of ZBTB24 or CDCA7 upregulates TRAIL & TRAIL-Rs in human T cells. • Blocking TRAIL-signaling abrogates apoptosis induced by ZBTB24/CDCA7-depletions. • ZBTB24 activates the expression of CDCA7 in T cells. • Overexpression of CDCA7 reverses the survival defects caused by ZBTB24-depletions. [ABSTRACT FROM AUTHOR]

The large ZBTB family comprises a diverse group of transcriptional factors. Several ZBTB proteins have emerged as critical factors that regulate the lineage commitment, differentiation, and function of lymphoid cells as well as many other developmental events. For instance, dysfunctions of ZBTB20 or ZBTB24 have been linked to multisystem failures in humans. Within the B-cell lineage, BCL6, ZBTB7A, ZBTB17, and ZBTB1 regulate the development/differentiation of B cells in both bone marrow and peripheral lymphoid organs, while ZBTB20 and ZBTB32 seem to mainly impact the maintenance of terminal plasma cells. Given the importance of B cells in the prevention and treatment of infectious or autoimmune disorders, we herein summarize the roles of seven ZBTB family members (BCL6, ZBTB7A, ZBTB17, ZBTB20, ZBTB32, ZBTB1, and ZBTB24) in the development, differentiation, and function of B cells as well as the underlying molecular mechanisms. [ABSTRACT FROM AUTHOR]

Immunodeficiency, centromeric instability and facial anomalies syndrome 2 (ICF2) is a rare autosomal recessive primary immunodeficiency disorder. So far, 27 patients have been reported. Here, we present three siblings with ICF2 due to a homozygous ZBTB24 gene mutation (c.1222 T>G, p. (Cys408Gly)). Immune deficiency in these patients ranged from late-onset combined immunodeficiency (CID) with severe respiratory tract infections and recurrent shingles to asymptomatic selective antibody deficiency. Evident clinical heterogeneity manifested despite a common genetic background, suggesting the pathogenic relevance of epigenetic modification. Immunological follow-up reveals a previously unidentified gradual depletion of B and CD4+ T cells in all three presented patients with transition of a common variable immunodeficiency (CVID)-like disease to late-onset-CID in one of them. Considering all previously published cases with ICF2, we identify inadequate antibody responses to vaccines and reduction in CD27+ memory B cells as prevalent immunological traits. High mortality among ICF2 patients (20%) together with the progressive course of immunodeficiency suggest that hematopoietic stem cell transplantation (HSCT) should be considered as a treatment option in due time.

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. [ABSTRACT FROM AUTHOR]

Chouery E, Abou-Ghoch J Corbani S, El Ali N, Korban R, Salem N, Castro C, Klayme S, Azoury-Abou Rjeily M, Khoury-Matar R, Debo G, Germanos-Haddad M, Delague V, Lefranc G, Mégarbané A. A novel deletion in ZBTB24 in a Lebanese family with immunodeficiency, centromeric instability, and facial anomalies syndrome type 2. The immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome is a rare autosomal recessive disease characterized by targeted chromosome breakage, directly related to a genomic methylation defect. It manifests with phenotypic and clinical variability, with the most consistent features being developmental delay, facial anomalies, cytogenetic defects and immunodeficiency with a reduction in serum immunoglobulin levels. From the molecular point of view, ICF syndrome was always divided into ICF type I (ICF1) and ICF type 2 (ICF2). Mutations in DNMT3B gene are responsible for ICF1, while mutations in ZBTB24 have been reported to be responsible for ICF2. In this study, we describe a Lebanese family with three ICF2 affected brothers. Sanger sequencing of the coding sequence of ZBTB24 gene was conducted and revealed a novel deletion: c.396_397delTA (p.His132Glnfs*19), resulting in a loss-of-function of the corresponding protein. ZBTB24 belongs to a large family of transcriptional factors and may be involved in DNA methylation of juxtacentromeric DNA. Detailed molecular and functional studies of the ZBTB24 and DNMT3B genes are needed to understand the pathophysiology of ICF syndrome. [ABSTRACT FROM AUTHOR]

The immunodeficiency, centromeric instability, facial anomalies (ICF) syndrome is an autosomal recessive disease presenting with immunodeficiency secondary to hypo- or agamma-globulinemia, developmental delay, and facial anomalies. Centromeric instability is the cytogenetic hallmark of the disorder which results from targeted chromosomal rearrangements related to a genomic methylation defect. We describe a patient carrying a homozygous mutation of the ZBTB24 gene, which has been recently shown to be responsible for ICF syndrome type 2. Our patient presented with intellectual disability, multiple café-au-lait spots, and a large cerebral arachnoidal cyst. Although laboratory signs of impaired immune function, such as reduced serum IgM were detected, our patient did not present clinical manifestations of immunodeficiency. Brain malformations abnormalities have not been reported so far in ICF syndrome and it can be speculated that ZBTB24 mutations may alter cerebral development. Nevertheless, we cannot rule out that the presence of the cerebral cyst in the patient is coincidental. In summary, our patient illustrates that clinical evidence of immunodeficiency is not a universal feature of ICF2 syndrome type 2 and suggests that brain malformations may be present in other ICF cases. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Recurrent spontaneous abortion (RSA) is a common complication during early gestation, which is associated with aberrant DNA methylation. Zinc Finger and BTB Domain Containing 24 (ZBTB24) plays a critical role in facilitating DNA methylation and cell proliferation. However, the regulatory role of ZBTB24 on trophoblast development in RSA remains unclear. In this study, ZBTB24 expression was compared between decidua tissues of RSA patients and induced abortion controls from a published dataset, which was further validated in placental villi tissues by RT-qPCR and Western blot. The roles of ZBTB24 in trophoblast proliferation, differentiation, and migration were investigated by functional assays after ZBTB24 knockdown or overexpression in HTR-8/SVneo cells. Our results showed that ZBTB24 expression was significantly decreased in RSA patients, and ZBTB24 expression level positively regulated cell viability, differentiation, and migration in HTR-8/SVneo cells. We further demonstrated that ZBTB24 modulated the expression of E-cadherin by altering the DNA methylation at the promoter region. Overall, the downregulation of ZBTB24 is implicated in RSA by inhibiting trophoblast proliferation, differentiation, and migration. Therefore, ZBTB24 may serve as a promising therapeutic target and diagnostic marker for RSA.

Immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome(s) are a group (ICF1 to ICF4) of autosomal recessive combined immunodeficiency disorders that may mimic common variable immunodeficiency (CVID) at initial presentation. Unlike CVID, autoimmune manifestations have been reported uncommonly in patients with ICF2. Herein we describe a new case of ICF2 with a novel ZBTB24 mutation and granulomatous hepatitis, with a literature review of all patients with ZBTB24 mutations. Post-neonatal hepatitis, reported in only 2 patients of ICF2 till date, was the presenting manifestation of the index child with ICF2. Evaluation revealed a homozygous mutation in ZBTB24 gene (c.433_434delGC, p.Ala145ProfsTer7). On literature review a total of 39 cases with ZBTB24 mutations reported till date were found, with two-thirds of reported patients being males. Respiratory tract infections and facial anomalies are commonest clinical features seen in more than 80 % of the patients. All patients who have immunoglobulin levels tested have at least 1 isotype decreased with decreased B cell number seen in at least one-third of patients. Decreased IgG and IgA levels are seen more frequently in patients with truncation mutations as compared to missense mutations. Candidiasis and Pneumocystis infections have been reported only in patients with truncation mutations. Facial features should be looked for in all patients presenting with hypogammaglobulinemia. Next generation sequencing should be considered in patients who have a CVID like presentation in early age with unusual manifestations. [ABSTRACT FROM AUTHOR]

Background: Immunodeficiency, centromeric instability and facial dysmorphism (ICF) syndrome is a rare autosomal recessive immune disorder presenting with hypogammaglobulinemia, developmental delay, and facial anomalies. The ICF type 1, type 2, type 3 and type 4 are characterized by mutations in DNMT3B, ZBTB24, CDCA7 or HELLS gene, respectively. This study aimed to present a comprehensive description of the clinical, immunologic and genetic features of patients with ICF syndrome., Methods: PubMed, Web of Science, and Scopus were searched systemically to find eligible studies., Results: Forty-eight studies with 118 ICF patients who met the inclusion criteria were included in our study. Among these patients, 60% reported with ICF-1, 30% with ICF-2, 4% with ICF-3, and 6% with ICF-4. The four most common symptoms reported in patients with ICF syndrome were: delay in motor development, low birth weight, chronic infections, and diarrhea. Intellectual disability and preterm birth among patients with ICF-2 and failure to thrive, sepsis and fungal infections among patients with ICF-1 were also more frequent. Moreover, the median levels of all three immunoglobulins (IgA, IgG, IgM) were markedly reduced within four types of ICF syndrome., Conclusion: The frequency of diagnosed patients with ICF syndrome has increased. Early diagnosis of ICF is important since immunoglobulin supplementation or allogeneic stem cell transplantation can improve the disease-free survival rate., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

DNA methylation is an epigenetic modification essential for normal mammalian development. Initially associated with gene silencing, more diverse roles for DNA methylation in the regulation of gene expression patterns are increasingly being recognized. Some of these insights come from studying the function of genes that are mutated in human diseases characterized by abnormal DNA methylation landscapes. The first disorder to be associated with congenital defects in DNA methylation was Immunodeficiency, Centromeric instability, Facial anomalies syndrome (ICF). The hallmark of this syndrome is hypomethylation of pericentromeric satellite repeats, with mutations in four genes: DNMT3B, ZBTB24, CDCA7 and HELLS, being linked to the disease. Here, we discuss recent progress in understanding the molecular interactions between these genes and consider current evidence for how aberrant DNA methylation may contribute to the abnormal phenotype present in ICF syndrome patients., (© 2019 The Author(s).)

Immunodeficiency, centromeric instability and facial anomalies syndrome 2 (ICF2) is a rare autosomal recessive primary immunodeficiency disorder. So far, 27 patients have been reported. Here, we present three siblings with ICF2 due to a homozygous ZBTB24 gene mutation (c.1222 T>G, p. (Cys408Gly)). Immune deficiency in these patients ranged from late-onset combined immunodeficiency (CID) with severe respiratory tract infections and recurrent shingles to asymptomatic selective antibody deficiency. Evident clinical heterogeneity manifested despite a common genetic background, suggesting the pathogenic relevance of epigenetic modification. Immunological follow-up reveals a previously unidentified gradual depletion of B and CD4+ T cells in all three presented patients with transition of a common variable immunodeficiency (CVID)-like disease to late-onset-CID in one of them. Considering all previously published cases with ICF2, we identify inadequate antibody responses to vaccines and reduction in CD27+ memory B cells as prevalent immunological traits. High mortality among ICF2 patients (20%) together with the progressive course of immunodeficiency suggest that hematopoietic stem cell transplantation (HSCT) should be considered as a treatment option in due time. [ABSTRACT FROM AUTHOR]

Background Immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is a rare, genetically heterogeneous, autosomal recessive disorder. Patients suffer from recurrent infections caused by reduced levels or absence of serum immunoglobulins. Genetically, 4 subtypes of ICF syndrome have been identified to date: ICF1 ( DNMT3B mutations), ICF2 ( ZBTB24 mutations), ICF3 ( CDCA7 mutations), and ICF4 ( HELLS mutations). Aim To study the mutation spectrum in ICF syndrome. Materials and methods Genetic studies were performed in peripheral blood lymphocyte DNA from suspected ICF patients and family members. Results We describe 7 ICF1 patients and 6 novel missense mutations in DNMT3B, affecting highly conserved residues in the catalytic domain. We also describe 5 new ICF2 patients, one of them carrying a homozygous deletion of the complete ZBTB24 locus. In a meta-analysis of all published ICF cases, we observed a gender bias in ICF2 with 79% male patients. Discussion The biallelic deletion of ZBTB24 provides strong support for the hypothesis that most ICF2 patients suffer from a ZBTB24 loss of function mechanism and confirms that complete absence of ZBTB24 is compatible with human life. This is in contrast to the observed early embryonic lethality in mice lacking functional Zbtb24. The observed gender bias seems to be restricted to ICF2 as it is not observed in the ICF1 cohort. Conclusion Our study expands the mutation spectrum in ICF syndrome and supports that DNMT3B and ZBTB24 are the most common disease genes. [ABSTRACT FROM AUTHOR]