Mutations in the TNFRSF1A, CEBPE and ELANE genes in a Congenital Cyclic Neutropenia Patient: A New Syndrome?

Abstract 11

Cyclic neutropenia (CyN) is a hematologic disorder in which blood cell counts particularly granulocytic neutrophil numbers show cycles at 21 day intervals. The majority of CyN patients (ca 90 %) harbor inherited mutations in the ELANE gene. Intriguingly, same ELANE mutations are present in two different hematologic syndromes: congenital as well as in cyclic neutropenias. It is unclear how mutation in the same gene cause congenital or cyclic neutropenia. We aimed to identify genes which are exclusively mutated in cyclic or in congenital neutropenia additionaly to the ELANE gene mutations. Recently, we found in congenital neutropenia patients additional to ELANE mutations inherited mutations in for example the G6PC3 gene or the HAX1 gene (Germeshausen, M., et al, Haematologica 2010). This suggests cooperating effects of different defective intracellular signaling pathways and excludes that mutated ELANE alone is responsible for the pathogenesis of congenital or cyclic neutropenia.

To identify gene mutations causing cyclic neutropenia in association with ELANE mutation, we performed whole genome sequencing using Complete Genomics technology (Complete Genomics. Inc, Mountain View, CA.) of a family with an affected CyN patient. The CyN patient harbors sporadic heterozygous ELANE mutation (c.761C>G p.W241L) and her family consists of a healthy brother and healthy parents. We identified a novel heterozygous point mutation in the tumor necrosis factor receptor superfamily, member 1A (TNFRSF1A) gene (c.664C>T; p.R121Q) in the affected patient and her mother. This mutation was confirmed by Sanger sequencing. The TNFRSF1A gene encodes p55 subunit of the TNFa receptor (TNFR1) and intriguingly this gene is known to be frequently mutated in patients with Tumour necrosis factor receptor-associated periodic fever syndrome (TRAPS), a disease clinically similar to cyclic neutropenia. TRAPS is an autosomal dominant disorder characterized by episodes of fever, inflammation and periodical changes in the neutrophil counts. Functional studies in patients with TRAPS described heterogenous effects of different TNFRSF1A mutations on the surface expression and PMA-induced clevage of TNFR1 on the neutrophilic granulocytes and monocytes. In our CyN patient we measured elevated mRNA levels of TNFR1 on neutrophils, in comparison to her healthy family members and unrelated healthy individuals. However, we detected diminished surface expression of the TNFR1 protein, but elevated PMA-induced receptor shedding in the affected CyN patient, in comparison to healthy individuals, as assessed by estimation of soluble TNFR1 in supernatants of PMA-stimulated neutrophils.

We also identified in the same patient and her father a second novel heterozygous point mutation in the CEBPE gene (c.636C>A; p.L155M), which was also confirmed by Sanger sequencing. This C>A substitution changes CTG to ATG creating a new start site for translation of a novel isoform of C/EBPε protein. C/EBPε is a myeloid-specific transcription factor playing an important role in granulopoiesis. Mutations in the CEBPE gene have been described in patients with neutrophil-specific granule deficiency (SGD).

In summary, we identified additional to the ELANE mutation two novel mutations in a CyN patient, one in the TNFRSF1A gene inherited from the mother, another in the CEBPE gene inherited from the father. Mutations in both genes are already described in patients with TRAPS (periodic fever syndrome) and granulocyte abnormalities, respectively. These mutations in association with the ELANE gene mutation may contribute to the pathogenesis of cyclic neutropenia in this patient. Whether the combination of these three mutations might be responsible for a subgroup of CyN patients remains to be investigated.