In 2001 we described X-linked neutropenia as a new syndrome of neutropenia caused by a gain-of-function type mutation of the WAS
gene (Devriendt, et al 2001
). Since, two additional isolated cases have been described, one with an I294T mutation and a second with an S272P mutation (Ancliff, et al 2006
). We here report a large family with XLN, in which the I294T mutation was identified. This family had previously been described in the literature as “a large kindred with congenital neutropenia and low serum immunoglobulin A” (Cryan, et al 1988
). This largest XLN kindred reported to date allowed us to describe the clinical, hematological and immunological phenotype of XLN in more detail.
Chronic neutropenia and monocytopenia are confirmed as prominent features of XLN, although some cases have values that fall within the low-normal range. This leads to a variable infectious phenotype which is not distinctive and overall clinically mild in view of the depth of neutropenia. The rate of infections does not correlate closely with the neutrophil count. Therefore, it seems reasonable to reserve the use of G-CSF for severe infections, so as to avoid the long-term risks of chronic G-CSF administration (Germeshausen, et al 2007
). The large number of affected males in this three-generation family allowed us to identify several, more subtle features of the phenotype of XLN: For instance, NK cell numbers, B-lymphocyte counts, platelet counts and levels of serum IgA were all subnormal or in the low-normal range. Importantly, the normal mean platelet volume allows to distinguish between X-linked neutropenia and X-linked thrombocytopenia, the attenuated phenotype of Wiskott-Aldrich syndrome with microplatelets. In contrast with 3/3 evaluated cases with L270P XLN, and two recently described cases with I294T and S272P XLN, an inverted CD4/CD8 ratio or elevated peripheral CD3+/CD8+ cell counts were not found in this family. Additional recent testing of cases IV.1 and IV.3 with L270P XLN did not reveal an inverted CD4/CD8 cell ratio either (unpublished data). Therefore, an inverted CD4/CD8 ratio does not seem an essential presenting feature of XLN. However, there was a tendency for decreased numbers of CD3+CD4+ helper T-cells, while the levels of CD3+CD8+ cytotoxic T-cells were better preserved.
The infectious pattern in XLN patients is not suggestive of a deficiency in T-cell mediated immunity. An elaborate immunological study was not performed in this family, although we attempted T-cell stimulation assays on frozen samples of 6 XLN patients (data not shown). Only in patient II.4, the recovery and condition of thawed cells was satisfactory for further testing. There was no clear deficit in T-cell production of cytokines in this patient, consistent with our previous findings in the L270P family. A possible explanation for the difference with classic Wiskott-Aldrich syndrome could be that only a truncated or absent WASP protein leads to deficient T-cell immunity. In contrast, full length WASP albeit mutated as in XLN or XLT remains compatible with preservation of a largely normal B-and T-cell function.
The thermal dissociation and actin-polymerisation studies allow to conclude that the I294T WAS mutation affects the normal function of WASP by a mechanism similar to L270P: that is, substitution of the non-polar isoleucine in position 294 by a hydrophilic threonine leads to disruption of the hydrophobic fold, loss of the auto-inhibited structure of inactive WASP and constitutive availability of the carboxy-terminal VCA-segment for actin-polymerisation.
Recent studies have partly elucidated the pathogenesis of neutropenia in XLN: I294T WASP causes hyperactivation and delocalisation of actin polymerisation, genomic instability and increased apoptosis (Moulding, et al 2007
). This could fit with the diagnosis of myelodysplastic syndrome (MDS) in one case (Ancliff, et al 2006
), and an eventual evolution towards MDS and acute myeloid leukaemia in two cases in the L270P family (Beel, et al 2006
). However, in the present kindred, no cases of MDS or leukaemia have occurred nor did available bone marrow and cytogenetic examinations reveal evidence for myelodysplasia or other myeloid malignancies.
X-chromosome inactivation studies, performed on sorted leukocyte populations from female I294T carriers of this family, did not reveal a consistent asymmetrical X-inactivation pattern. This was unexpected as previous studies on unfractionated leucocytes in the L270P family had shown asymmetrical X-inactivation in 2/2 tested carrier females (Devriendt, et al 2001
). In addition, three carriers with S272P and one carrier with I294T WAS
also displayed asymmetrical X-inactivation in haematological cells. (Ancliff, et al 2006
). These seemingly conflicting results could reflect that activating mutations in the GBD of WASP do not provide a sufficiently strong signal to consistently result in skewed X-chromosome inactivation. Indeed, carriers of XLT may have random X-inactivation in haematological cells, while carriers of inactivating WAS
mutations, leading to Wiskott-Aldrich Syndrome, uniformly show non-random inactivation of the X-chromosome harbouring mutant WAS
(Puck, et al 1990
; De Saint-Basile, et al 1991
; De Saint-Basile, et al 1996
). Alternatively, Carrel and Willard have recently shown that WAS
belongs to the small proportion of genes that can be variably expressed from the inactivated X-chromosome (Carrel, et al 2005
), which could also explain the lack of skewing in hematopoietic cells of this family and the mild neutropenia that is observed in carrier females. Finally, other background genetic factors may contribute to determine the ratio of X-chromosome inactivation (Puck, et al 1998
In conclusion, the data from this large pedigree with the I294T WAS
mutation provide important, new and independent genetic evidence that mutations disrupting the auto-inhibitory GBD domain of WASP are the cause of XLN. Clinically, the severity of the neutropenia in I294T XLN does not seem to translate into an extreme susceptibility to infections. NK cell numbers, platelets and IgA levels in XLN are consistently below normal or in the low-normal range. There is a tendency for decreased numbers of CD4, but reversed CD4/CD8 ratio’s, as previously described by us and others (Ancliff, et al 2006
, Devriendt, et al 2001
), do not seem to be invariably present. Unexpectedly for an X-linked disease, female carriers of XLN have intermediate neutrophil, NK cell and platelet numbers and intermediate levels of IgA. To date, no affected members of this family have developed malignancies of myelopoiesis. However, as most affected males are younger than in the Belgian family, it is advisable that they are followed up closely. In particular long term G-CSF usage may predispose to CSF3R mutations, and this is highly predictive for malignant transformation (Germeshausen, et al 2007
). Therefore, it might be reasonable to limit the use of G-CSF to episodes of severe infection and chronic use to merely correct neutrophil count should be discouraged.
Finally, further investigation is needed to reveal how L270P and I294T WASP mutations lead to XLN.