Novel ZBTB24 mutation associated with immunodeficiency, centromere instability, and facial anomalies type 2 syndrome identified in a patient with very early onset inflammatory bowel disease

Abstract

Background

Very early onset inflammatory bowel disease, diagnosed in children ≤5 years of age, can be the initial presentation of some primary immunodeficiencies.

Methods

Here we describe a 17 month old boy with recurrent infections, growth failure, facial anomalies, and inflammatory bowel disease. Immune evaluation, whole exome sequencing, karyotyping, and methylation array were performed to evaluate this child’s constellation of symptoms and exam findings.

Results

Whole exome sequencing revealed that the child was homozygous for a novel variant in ZBTB24, the gene associated with immunodeficiency, centromere instability, and facial anomalies type 2 syndrome (ICF2).

Conclusion

This describes the first case of inflammatory bowel disease associated with ICF2 in a child with a novel disease-causing mutation in ZBTB24 found on whole exome sequencing.

Introduction

The diverse spectrum of primary immunodeficiencies includes more than 250 rare chronic diseases that affect both innate and adaptive immunity. Gastrointestinal manifestations can be the primary presentation of immune deficiencies, including diarrhea, malabsorption, abdominal pain, and inflammatory bowel disease.¹ Gastrointestinal tract involvement and symptoms in immunodeficiency are not surprising as the intestinal tract contains the largest mass of lymphoid tissue, and the mucosa is at the interface of host-microbe interactions. The mucosal immune system requires a tightly controlled balance between tolerance of commensal microbes and detection and response against pathogens. Rare monogenic defects in immune pathways have been identified in very young patients with inflammatory bowel disease, known as very early onset inflammatory bowel disease (VEO-IBD), diagnosed in children who present with symptoms by the age of 5.^2,3 VEO-IBD is a heterogeneous disorder with a subset of patients who present with severe disease, often refractory to conventional medical therapies compared to older onset IBD. The increased utilization of whole exome sequencing (WES) has identified several novel variants in immune pathways that are associated with this phenotype and there are now over 50 monogenic defects detected in patients with severe VEO-IBD.^4,5 Here we present a novel mutation in ZBTB24, the gene responsible for Immunodeficiency, Centromere instability, and Facial anomalies Type 2 syndrome (ICF2, OMIM #614064), detected by WES in a child with VEO-IBD. This is the first report of inflammatory bowel disease associated with this syndrome.

Methods

A 17-month old boy initially presented with several months of poor weight gain and linear growth, recurrent fevers, constipation, and intermittent blood in his stool. Pertinent family history included multiple layers of consanguinity as well as Crohn disease in his maternal grandmother. His physical exam was notable for hypertelorism, epicanthal folds, low set posteriorly rotated ears, developmental delay, and perianal fistula on rectal exam. Colonoscopy revealed multiple ulcerations in descending colon and rectum. The histology showed active chronic colitis with crypt architectural distortion. He was initially treated with azathioprine, corticosteroids, mesalamine enemas, and metronidazole and had resolution of his symptoms and fistula within 6 months. He subsequently developed serious infections, including multiple episodes of febrile pneumonia requiring hospitalizations and treatment with intravenous antibiotics. Additionally, he had two episodes of acute otitis media but no sinusitis or cellulitis, and his growth remained poor.

He underwent clinical immune work up including evaluation of his B cells, T cells, NK cells, immunoglobulins, and vaccine titers. Under protocol 2014-010826 approved by the Children’s Hospital of Philadelphia Institutional Review Board, genomic DNA was obtained from the child and his parents. The Agilent SureSelect Clinical Research Exome kit, targeting the coding exonic regions of the genome, was used for library preparation. Sequencing was performed using the Illumina HiSeq 2000 with 100bp paired-end reads. Sequence data was assembled and aligned to the reference human genome browser GRCh37/UCSC hg19 using Novoalign (V3.03.01; http://www.novocraft.com). Sequence variants were identified using GATK’s best practices and annotated for allele frequencies using the reference databases (1000 Genomes Project, ESP and ExAC) and known disease variants using the (Human Genome Mutation Database and NCBI’s ClinVar).^6,7 Analysis for the patient included evaluating variants that are de novo, compound heterozygous, homozygous, and X linked. Identified variants were filtered for rare (MAF < 1%) or novel missense, nonsense and frameshift mutations, and restricted to genes involved in primary immunodeficiency and inflammatory bowel disease based on the clinical phenotype.

DNA obtained from the patient, his parents, and a patient with VEO-IBD without a mutation in ZBTB24 were bisulfite converted and hybridized to the Illumina HumanMethylation450 BeadChip array using the Infinium HD Methylation protocol (Illumina, San Diego, CA, USA). Beta values representing the level of methylation of a probe were obtained using Genome Studio. β-values range from 0 (completely unmethylated) to 1 (methylated). Data was processed using the Illumina Methylation Analyzer(IMA) package in R.⁸ Briefly, sites containing missing values were removed, peak correction was performed, and the data was quantile normalized. Probes with a detection p-value > 0.05 and those containing single nucleotide polymorphisms were removed from further analysis. A total of 392,222 probes passed quality control. Alpha satellite region coordinates were obtained from the USCS Genome Browser to identify probes within these regions. Differentially methylated probes were identified as those with a β-value difference >|0.2| in the patient compared to the two healthy parents.

Results

Immune work up revealed a combined immunodeficiency including total hypogammaglobulinemia, decreased production and maturation of B cells, decreased natural killer cells, and poor titer response to vaccines (Table 1).

Table 1.

Immune profile of subject demonstrating hypogammaglobulinemia, poor vaccine titer response, absent switch memory B cells, normal naïve and memory T cells, and low number of natural killer cells.

Lab Test	Value	Normal range
Immunoglobulin Evaluation:
Immunoglobulin A	48 mg/dL	49–336 mg/dL
Immunoglobulin E	<4 IU/mL	0–60 IU/mL
Immunoglobulin G	30 mg/dL	539–1597 mg/dL
Immunoglobulin M	17 mg/dL	50–194 mg/dL
Anti-Diphtheria Antibodies	0.01	>0.10 IU/mL
Pneumococcal IgG Antibodies (14 serotypes)	</=0.11 ug/mL
Total WBC count	11.5 THOU/uL	5.14–13.38 THOU/uL
B Cells:
CD19+	15.2%	10–31%
CD19+/CD27+/IgM−	0%	3.9 – 30.7 % of CD19
CD19+/CD27+/IgD−	0.1%	4.6–33.7 % of CD19
Natural Killer Cells:
CD3−/CD16(B73.1) &/OR CD56+	2.6%	4–26%
T Cells:
CD3+	81.8%	55–78%
CD3+/CD4+	48.1%	27–53%
CD3+/CD8+	34.1%	19–34%
CD4+/CD45RA+	25.0%	12.5–42.2%
CD4+/CD45RO+	24.2%	10.1–27.9%

WES detected a potential causative homozygous frameshift mutation, c.1492-1493delCA: p.Gln498ValfsX15, in ZBTB24, which is involved in B cell differentiation and has previously been associated with immunodeficiency, centromere instability, and facial anomalies, type 2 syndrome, characterized by facial anomalies, developmental delay, and recurrent respiratory and gastrointestinal infections due to a multifactorial immunodeficiency. The patient’s mother and father are healthy and heterozygous for the same mutation. Karyotype revealed chromosomal changes due to centromere instability in 1q, 9q, and 16q, including heterochromatin decondensation and stretching, multiradial configurations, and deletions, which are unique features of ICF and confirmed this diagnosis. (Figure 1)

Figure 1. Abnormal karyotype findings in this patient with ZBTB24 mutation demonstrating.

a) stretching and decondensation of heterochromatin region,

b) deletion of chromosome 1q, 16q, and 16, and

c) triradial formations of chromosome 16q

Upon this diagnosis, further evaluation of his intestinal biopsies revealed a hypocellular lamina propria in the duodenum that had minimal CD79a histochemical staining of a few scattered plasma cells and absent immunohistochemical staining for both IgA and IgG producing plasma cells in comparison with another VEO-IBD patient without the ZBTB24 mutation. (Figures 2a and 2b).

Figure 2.

Figure 2A: Duodenal histopathology of patient with VEO-IBD but without ZBTB24 mutation with CD79a histochemical staining highlighting abundant plasma cells in lamina propria

Figure 2B: Duodenal histopathology of this patient with ZBTB24 mutation with CD79a histochemical staining showing few scattered plasma cells in lamina propria

Additionally, a homozygous variant of unknown significance in IL7R (c.419G>A:p.Arg140Gln) was detected by WES in the child, and confirmed in heterozygosity in both parents. This gene has been previously associated with a severe form of combined immunodeficiency that is defined by the absence of mature T cells and the presence of B cells and NK cells. These findings were not consistent with this patient’s immune profile, and the variant was not novel. There were no variants in the genes known to be associated with the adult onset form of inflammatory bowel disease.

Prior work described distinctive methylation defects in patients with ICF1, thus methylation analyses were performed in this patient.⁹ A total of 15 probes overlapped alpha satellite regions obtained from the UCSC Genome Browser, including 8 probes on chromosome 1 and none on chromosomes 9 and 16. Three of the 15 probes (20%) were unmethylated in the patient compared to his healthy parents, including 1 probe on each of chromosomes 7, 10 and 18. None of the probes were hypermethylated in the patient compared to his parents. Despite the small number of alpha satellite regions represented, there was a significant representation of hypomethylated probes in the patient (p=4×10⁻³). A similar analysis in a VEO-IBD patient without any mutations in ZBTB24 did not result in any differentially methylated probes within the alpha satellite regions.

Discussion

Monogenic defects of primary immunodeficiencies involving B and T cell abnormalities, phagocyte defects, epithelial barrier defects, and autoimmunity can present as very early onset inflammatory bowel disease.¹⁰ In this patient with inflammatory bowel disease and recurrent infections, the immunologic work up was concerning for hypogammaglobulinemia, and whole exome sequencing revealed a homozygous novel frameshift variant in ZBTB24, consistent with a diagnosis of Immunodeficiency, Centromere instability, and Facial anomalies syndrome, type 2. Up to 13 homozygous and compound heterozygous mutations of ZBTB24 have been described in patients with this syndrome, characterized by fragile heterochromatin resulting in unstable chromosomes, which is associated with immunodeficiency, most commonly hypo- or agammaglobulinemia, facial anomalies including hypertelorism, flat nasal bridge, epicanthal folds, micrognathia, and tongue protrusion, and variable intellectual disability.^11,12 Inflammatory bowel disease confirmed by endoscopic biopsy has not previously been reported with ICF2 syndrome, but IBD has been associated with other humoral immunodeficiencies defined by hypo- or agammaglobulinemia. This association has led to the use of intravenous immunoglobulin as a therapy for IBD in some refractory cases.

There are currently five subtypes of ICF syndrome, all phenotypically very similar, and ICF types 1–4 compose 80% of all cases. About half of ICF cases are due to mutations in DNA methyltransferase 3B gene (DNMT3B), which is involved in de novo DNA methylation resulting in ICF1, and 30% of all cases are due to mutations in ZBTB24, which encodes a protein involved in the methylation of juxtacentromeric DNA resulting in ICF2. Recent advances in the investigation of those cases of ICF without known gene defects, have identified cell division cycle associated 7 (CDCA7) and helicase, lymphoid specific (HELLS) as the causative genes in ICF3 and 4.¹³ The remaining cases with thus far unknown genetic mutations are known as ICFX.

ZBTB24 is a member of the zinc binding and BTB-domain binding family of transcription factors, some of which are involved in hematopoietic development, differentiation, and function. Interestingly, the immune dysregulation in ZBTB24 deficiency resembles the phenotype of ZBTB27 mutations. ZBTB27, also known as BCL-6, is necessary for germinal center structures, generation of B cells, and production of antibodies. Immunophenotyping of patients with ZBTB24 mutations, including our patient, reveals similar defects in circulating memory B cells. Liang et al demonstrated that ZBTB24 is a novel transcription factor involved in human B cell proliferation and differentiation.¹⁴ These functions are mediated through IRF-4 and BLIMP-1. This essential role in B cells secondary to the reduced proliferation of germinal center B cells results in the phenotype of reduced serum immunoglobulins and memory B cells as seen in our patient.

Previously reported nonsense mutations in ZBTB24 were associated to hypomethylation of centromeric alpha satellites and juxtacentromeric satellite repeats. This results in the characteristic multibranching of long arms of chromosomes 1, 9, and 16, causing radial chromosomes. A cause for the structural abnormalities only in these chromosomes has yet to be elucidated. For this patient, we demonstrated that there was a significantly increased number of hypomethylation probes in the alpha satellite regions compared to his healthy parents and another child with VEO-IBD without mutations in ZBTB24. The association of this defect with inflammatory bowel disease remains to be explained and warrants further studies.

This is the first report of very early onset inflammatory bowel disease in association with homozygosity for a novel mutation in ZBTB24, consistent with a diagnosis of immunodeficiency, centromere instability, and facial anomalies, type 2 syndrome. Primary immunodeficiencies can present with phenotypic characteristics of VEO-IBD as well as other autoimmune disorders. The intestinal phenotype of IBD is likely secondary to the overall immune dysregulation in this patient. Here whole exome sequencing together with immunophenotyping detected a novel variant in a known immunodeficiency gene that presented as recurrent pneumonia and inflammatory bowel disease. Following the identification of the ZBTB24 gene defect and clinical finding of hypogammaglobulinemia, the patient was treated with intravenous immunoglobulin monthly and was able to successfully induce and maintain remission as well as avoid further recurrent infections during the period of follow up.

List of Abbreviations

IBD

inflammatory bowel disease

ICF2

Immunodeficiency, centromere instability, and facial anomalies syndrome, type 2

NK cells

natural killer cells

VEO-IBD

very early onset inflammatory bowel disease