Immunophenotype, Karyotype and Molecular Findings in a Case of ICF Syndrome

Dear Editor

Immunodeficiency, centromere instability and facial anomalies (ICF) syndrome is a rare type of primary immunodeficiency disorder (PID) classified under combined immunodeficiencies with syndromic features in the IUIS classification [1]. Based on the underlying gene defect, four distinct subtypes of ICF syndrome have been characterized: ICF1 (DNMT3B), ICF2 (ZBTB24), ICF3 (CDCA7) and ICF4 (HELLS) [2, 3]. All four subtypes have an autosomal recessive inheritance with a severe immunodeficiency phenotype caused by reduced levels or absence of serum immunoglobulins. Apart from immunodeficiency and facial dysmorphism, the disease is characterised by the characteristic karyotype abnormalities because of the centromere instability. Following phytohemagglutinin (PHA) stimulation, aberrant multiradial configurations are seen with chromosomes 1, 9, and 16. The combination of a defective humoral immune response with normal or reduced B cell and T cell counts, facial anomalies and cytogenetic multiradial configurations is typical for all ICF syndrome patients [4].

Case Details

A 1 year old male child presented with complaints of fever, cough, and rapid breathing. He was admitted and intubated in view of an impending respiratory failure. The child was born of non-consanguineous marriage. He had episodes of focal encephalitis, gastroenteritis, and bronchopneumonia at 5, 6 and 8 months of age respectively. On examination, his abdomen was distended, and his liver was palpable 4 cm below the right costal margin. No facial dysmorphism or developmental delay was documented. Laboratory workup showed pan hypogammaglobulinemia and elevated C-Reactive protein (CRP). Blood culture showed pseudomonas growth while his hemoglobin and white blood cell counts were 6.0 gm/dL (3.72 mmol/L) and 3.3 × 10⁹/L respectively. Based on the history of recurrent infections, failure to thrive and hypogammaglobulinemia, the child was diagnosed with PID, probably severe combined immunodeficiency [5]. (Supplement file S1) Peripheral blood sample was sent for lymphocyte subset evaluation by flow cytometry and mutation analysis by next generation sequencing (NGS). Subset analysis showed that, despite the hypogammaglobulinemia, B cells constituted 55.2% of the total lymphocytes while the T cells and NK cells constituted 44.1% and 1.5% of the lymphocytes respectively. The T cells were predominantly CD4 + ve (84.4%) and showed naive and central memory phenotype which was consistent with the age of the child. Almost the entire B cell population (99.3%) had a pre-germinal centre phenotype (CD27-ve). The percentages of different T cell and B cell subsets are as shown in Fig. 1a.

Fig. 1.

a Immunophenotyping density plots showing the results of subset evaluation with a single tube 13-antibody panel acquired in DxFlex flow cytometer (Beckman Coulter). B cells constituted 55.2% of the gated lymphocytes and showed a pre-germinal centre CD27-ve phenotype (CM central memory, EM effector memory, TD terminally differentiated, GC germinal centre), b G banded chromosomes showing multi-branched configurations involving chromosome 1

The next generation sequencing protocol and the subsequent bioinformatics used for the characterization of variants have been described in detail earlier [5]. A homozygous missense variant, c.2003C > T was identified in exon 19 of DNMT3B (Refseq ID: NM_006892.4) which resulted in the substitution of threonine by isoleucine at codon 668 (p.Thr668Ile). The variant was predicted to be pathogenic based on the ACMG guidelines for the interpretation of germline variants and has been reported earlier in patients with ICF syndrome. The variant lies in the catalytic methyltransferase domain of DNMT3B. Following the genetic diagnosis, karyotyping was done to confirm the cellular phenotype. Metaphases obtained from PHA-stimulated peripheral blood cultures (72 h) showed multiradial configurations characteristic of ICF. (Fig. 1c) The child is currently worked up for hematopoietic stem cell transplantation.

Identifying the underlying pathogenic variant provided the option of prenatal diagnosis in the family. Chorionic villus sampling was done from the mother when she was 15 weeks pregnant. Primers were designed targeting exon 19 of DNMT3B and Sanger sequencing was done to screen for the mutation identified in the proband. Sequencing showed that the foetus did not harbour the mutation identified in the proband.

ICF syndrome is very rare with few published case reports and case series. Nearly half of the patients carry mutations in the DNMT3B gene located on chromosome 20 [3]. Majority of the DNMT3B mutations described in the literature cluster in the highly conserved catalytic domain of DNMT3B. Patients usually present with hypogammaglobulinemia resulting in recurrent infections. Subset analysis in the child revealed that > 99% of the B cells showed an unswitched (IgM/IgD + ve, CD27-ve) naive phenotype. This concurred with earlier studies which had shown that although the B cell absolute counts in ICF patients could be normal, they contain an increased proportion of immature or unswitched cells, show a lack of CD27 + ve memory cells and are more prone to undergo apoptosis upon in vitro activation [6]. The T cell count is generally within normal limits in ICF patients. However, opportunistic infections are common in ICF patients suggesting a functional T-cell defect. The patients typically show a multi radial configuration in karyotyping analysis involving one or more of chromosomes 1,9 and 16. These chromosomes are normally heavily methylated at cytosine residues which is essential for the organization and stabilization of heterochromatin. The mutations result in defective methylation at juxta centromeric regions of chromosomes resulting in the stretching of the centromeric heterochromatin visible as multiradial configurations [7]. The report highlights the importance of mutation screening and the role of NGS in establishing the diagnosis of this rare entity. Having a genetic confirmation of the diagnosis helps in taking important therapeutic decisions such as stem cell transplantation, genetic counselling and prenatal diagnosis.

Supplementary Information

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Declarations

Conflict of interest

The authors have no competing interests. All the authors declare that they have no involvement in or affiliations with any organization or entity with any interest, financial or otherwise in the subject matter or materials discussed in this manuscript.

Ethical Standard

This is a retrospective analysis using DNA extracted from EDTA blood sample that has been collected for routine molecular diagnostic procedures in patients with primary immunodeficiency in our institution. No additional information was gathered, nor sample collected from the patient for the purpose of this study. All procedures performed in this study were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments.