Written by 14:18 Van Casus naar Thesis

Van Casus naar Thesis

Author: Marin Bont, 3e jaars geneeskunde student CRU+


The US center of disease control and prevention (CDC) reported a discovery of 21 HIV-negative persons with unexplained CD4 T-lymphocyte depletion,  this led to a new syndrome: Idiopathic lymphocytopenia (ICL). The diagnosis is based on: <300 CD4 cells/ µL or <20% CD4/ total T cells, >2 times CD4 lymphopenia separated by 6 weeks, No evidence of HIV infection, any other disease or therapy explaining the lymphopenia (1). The syndrome affects both children and adults with symptoms ranging from life threatening opportunistic infections to asymptomatic (2).  The pathogenesis of ICL remains largely unknown, there are three causal mutations for ICL, indicating that ICL is a heterogeneous disease. There is no consensus on the need for antibiotic prophylaxis for patients with ICL.

Case presentation

Two siblings of unrelated parents were diagnosed with ICL. The older of the two boys presented at the Wilhelmina Children’s Hospital in March 2011 at the age of 7 with recurrent skin infection, pustulosis and impetigo vulgaris(3). Lymphocyte counts showed a consistent severe CD4+ T cell lymphopenia (126-250/mm3) without apparent cause.  The younger sibling also showed CD4 lymphopenia and was diagnosed with ICL, due to the low prevalence of the syndrome a genetic cause for the ICL seemed likely.

Primary outcome

These cases created a unique opportunity to identify a novel disease causing mutation in these patients. Based on the inheritance pattern, we hypothesized that a compound heterozygous, a autosomal recessive or a X-linked mutation caused ICL in these patients.

In order to identify mutations, the information-containing part of the hereditary material – the exome – is examined, The procedure was as follows: heparinized venous blood was collected from parents and siblings, afterwards peripheral blood mononuclear cells (PBMCs) were isolated. PBMCs from cord blood were obtained from the obstetrics department. Human total thymus from the patients were processed and thymocytes were viably frozen. Samples were thawed for mRNA isolation. The neuroblastoma cell line SH-SY5Y was used as a positive control. Genomic DNA was isolated from PBMCs from parents and patients. The exome of the samples were sequenced, and afterwards we calculated the scores to predict the probability of damaging mutations. Exome results were tested for a recessive single gene compound heterozygous, meaning two different mutations, one from each parent, in the same gene. 


There were 4744 variants identified in 3495 genes, after filtering there were 10 gene candidates. After further filtering and exclusion one variant was considered as the mutation responsible for ICL: UNC80. The mutation seen in our patients may be responsible for altering the bindings of UNC80 to another unidentified ion channel in T-cells. It is possible that UNC80 prolongs T-cell survival through increased intra-cellular calcium, which promotes T-cel survival. We hypothesized about two possible mechanisms of UNC80 involvement in T-cell survival:  either through voltage gated sodium channels (VGSC) or through voltage gated calcium channels (Cav). The importance of voltage gated sodium channels (VGSC) in positive selection of T cells has been described previously (4). 

Figuur 1. Voltage gated sodium channel (VGSC), T-cell receptor (TCR), voltage gated calcium channel (Cav), endoplasmatic reticulum (ER).

The expression of UNC80 was only shown in the positive control neuroblastoma cell line SH-SY5Y, not in the PBMCs or thymocytes. 


To examine whether UNC80 may be involved in this process the expression of UNC80 was measured in thymocytes and PMBCs. Since we did not find indications for UNC80 expression in either PBMCs or thymocytes it is unlikely to think that UNC80 has a function in CD4 homeostasis. However, if the mutation is restricted to thymus cells and results in reduced thymic output, is it possible that the mutation goes unnoticed in adults due to reduced contribution of the thymus in CD4 homeostasis in adults? We concluded this to be unlikely and ended our one-year journey to discover a novel gene related to ICL.


  1. SmartFlare™ RNA Detection Probes: Principles, protocols and troubleshooting
  2. Ahmad DS, Esmadi M, Steinmann WC. Idiopathic 
  3.  Lymphocytopenia: Spectrum of opportunistic infections, malignancies, and autoimmune diseases. Avicenna J Med. 2013;3(2):37-47.
  4. A healthy 7 year old boy with hardly any CD4+ T lymphocytes due to increased apoptosis combined with an indication of diminished thymic output. Marjolein Quaak, Louis Bont, Kiki Tesselaar (Unpublished)
  5.  Lo WL, Donermeyer DL, Allen PM. A voltage-gated sodium channel is essential for the positive selection of CD4(+) T cells. Nat Immunol. 2012;13(9):880-7.
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