The term “reticular dysgenesis” (RD), was coined in 1959 by de Vall and Seyneheve1
and relates to the histological findings in primary and secondary lymphohaematopoietic organs, where the scarcity of cells highlights the prominent reticular tissue framework. The lack of polymorphonuclear neutrophils (PMNs) in affected patients is responsible for the occurrence of severe infections earlier than is usually observed in other forms of SCID2,3
. RD-associated neutropenia is characterized by lack of responsiveness to granulocyte-colony stimulating factor (G-CSF)4
. The only available treatment for RD is allogeneic haematopoietic stem cell transplantation (HSCT), which formally demonstrates that the inherited defect is cellular and not micro-environmental5
In order to determine the molecular genetic basis of RD, we took advantage of three separate consanguineous kindreds (family A, B & C, ). A genome-wide linkage scan generated a significant, multipoint LOD-score of 4.47 in a single region located on the short arm of chromosome 1 (1p31–p34). The map was then refined by genotyping 17 additional microsatellite markers in this region. The 90% confidence interval (CI) of the region of interest covered ~ 4 Mbs, stretching from 30.89 to 34.79 Mb (). Interestingly, two patients originating from the same geographical area (P1 and P2) were found to be homozygous for the same haplotype in the region between 32.8 and 34.79 Mb (including 18 SNPs and 8 microsatellite markers), which strongly suggested a founder effect in these two families and reduced the prime region of interest to ~ 2 Mbs. The prime region of interest was then fully sequenced (Supplementary Table 1
). Patients from the three consanguineous families (P1, P2 and P3) were found to be homozygous for mutations in the adenylate kinase 2 (AK2
)-encoding gene. Similarly, we found AK2
mutations in four additional available RD patients born to non consanguineous parents ().
AK2 gene mutations in seven patients with reticular dysgenesis
gene encodes two types of mRNA (expressed via an alternative splicing mechanism): AK2A
(6 exons) and AK2B
which includes an additional seventh exon located in the 3′ part of the gene. AK2A
encodes, respectively, a 239-amino-acid (aa) and a 232-aa protein6
. Homozygous mutations were prevalent among the defects found in the seven RD patients analyzed (): 3 patients (P1, P2 and P5) showed homozygous missense mutations of highly conserved amino acid residues (Supplementary Fig. 1
), two patients (P3 and P6) carried homozygous deletion (1 bp- and ~5 kb, respectively) that generate a premature stop codon and in one case (P7) a homozygous nonsense mutation was detected. P4 was found to be a compound heterozygote with a missense mutation and an exon 2 deletion (). All mutations are located in different key domains of the AK2 protein () and are thus expected to severely hamper its stability and/or function. In all cases, the parents were found to be heterozygous for the mutated AK2 allele and the healthy siblings that we tested were heterozygous or homozygous for the wild-type allele ().
is expressed in the mitochondrial intermembrane space in a variety of tissues such as the liver, kidney, spleen and heart 6
. We assessed the expression of the AK2
gene in fibroblasts (either primary cultures or SV40-transformed cells) obtained from patients P1, P3, P5 and P6 and in a B-EBV cell line from patient P2. Using Western blot analysis, no AK2 protein was detected in the fibroblasts from P1, P3 and P6 (). The AK2 protein was detected in the B-EBV cell line and in fibroblasts derived from P2 and P5, respectively, but at markedly lower levels than in control cells (). In contrast to the AK2 protein, AK2
mRNA was detected by RT-PCR at levels comparable to those found in control cell lines in all patient samples analyzed (). These data strongly suggest that RD results from loss-of-function mutations in the AK2
Gene and protein expression analysis of AK2 in cell lines derived from RD patients
Analysis of the immunological characteristics of RD patients showed that the profound neutropenia was invariably associated with an equally profound T- and natural killer (NK) cell lymphopenia, whilst the B cell lineage was variably affected (). The circulating monocyte count was normal or above normal, whereas red blood cell and platelet counts were normal or slightly diminished, as also observed by Levinsky RJ et al7
. Morphological examination of the bone marrow revealed a profound block in granulopoiesis, with no detectable cells beyond the myelocyte stage, thus indicating that RD defect corresponds to a selective developmental arrest along T, NK lymphoid and granulocytic myeloid pathways. In semi-solid medium, CD34+
cells isolated from the BM of P2 did not generate granulocytic colonies and only a few myeloblasts and erythroblasts were observed before and after culture (Supplementary Fig. 2a
). In patient CD34+ cells cultured in the presence of SCF, FLT-3L and GM-CSF or G-CSF, CD15+
neutrophil cell counts were two and four times lower, respectively, than in control cord blood (CB) cell cultures (Supplementary Fig. 2b
) suggesting a potential role for AK2 in the G-CSF R mediated myeloid differentiation. This is in agreement with the G-CSF refractory neutropenia observed in RD patients.
Haematological and Immunological characteristics of the patients
As a first step towards elucidating the function of AK2 in haematopoiesis, we performed an RT-PCR AK2 gene expression profile analysis that revealed significant AK2
upregulation in the various haematopoietic subsets as compared to a fibroblast cell line (Supplementary Fig. 3
). These results fit with a differential and specific role of AK2
in different cell types.
In order to prove unequivocally that AK2
mutations were responsible for the profound neutropenia, we set out to restore myeloid differentiation in vitro
by transducing bone-marrow CD34+
cells from RD patients with an AK2
A+B cDNA-encoding lentivirus. Granulocyte/granulocyte-monocyte (G/GM) colonies derived from BM cells of P3 and transduced with a control GFP vector were characterized by the presence of immature myeloblasts, promyelocytes, myelocytes and very few mature polynucleated cells. In contrast, G/GM colonies derived from BM cells transduced with AK2
A+B+GFP contained around 46% of mature myeloid cells, metamyelocytes and polymorphonuclear cells () exhibiting the unambiguous characteristics of mature myeloid cells. These mature granulocytes were very similar to those observed in colonies cultured from control healthy cord blood cells (). We confirmed these morphological observations by flow cytometry analysis () and showed that the CD15+
cell count was 3.7-fold higher in the AK2A+B+GFP condition than in the GFP condition (). Furthermore, the overall count of GFP+
complemented cells in the G/GM colonies was 53-fold higher than that of GFP+
cells isolated from non-complemented BM cells (). Restoration of neutrophil differentiation was confirmed in BM cell from P6 (Supplementary Fig. 4
). These data demonstrate that AK2
complementation corrects the defective granulopoiesis in RD.
Complementation of the neutrophil differentiation defect by restoration of AK2 expression
In order to confirm the specific role of AK2 in neutrophil differentiation, we have developed a RNA interference strategy through lentiviral-mediated gene transfer of AK2 short hairpin RNAs (shAK2), using the previously published shAK2 target sequence 8
. The downregulation of AK2 expression in human CD34+
cell induced a 17 to 27-fold reduction in myeloid cells and granulocyte precursors associated with a profound arrest in neutrophil differentiation as determined by counting of CD15+
cells and by morphological examination (Supplementary Fig. 5
). Conversely, no decrease in the proliferation rate wa observed in human primary fibroblasts transduced with the same AK2 shRNA lentivirus vector (data not shown).
In order to investigate the pathogenic process underlying the hearing impairment observed in all 7 RD patients, we analyzed the distribution of AK2 in the mouse inner ear by immunohistolabelling. AK2 expression could not be detected in the vestibule, at any developmental stage (). In contrast, the assesment of the cochlea revealed an intense AK2 staining observed uniquely in the stria vascularis (SV) at post-natal day 7 but not at birth (). Co-immunolabelling of AK2 and isolectin showed that AK2 is present within the lumen of the SV capillaries (), thus suggesting that it could be here functioning as an ecto-enzyme. Notably, AK2 could not be detected in the capillaries or vessels of the adjacent connective tissue ().
AK2 distribution in the mouse inner ear
Our data provide evidence that recessive mutations of the human AK2
gene are responsible for RD- a very rare form of SCID characterized by a profound impairment of the lymphomyeloid compartment associated with deafness. AK2
plays a major role during development, as shown in Drosophila
−/− larvae are not viable9
. This enzyme regulates the homeostasis of cellular adenine nucleotides by converting ADP into ATP and AMP 9
and thus may be critical in specific key subcellular or extracellular compartments. Our observations in the inner ear suggest a new function for AK2 in the SV. Indeed, failure of the SV to produce the endocochlear potential or secrete K+
in the endolymph results in hearing impairment10,11
. Moreover, serum ADP is thought to be one of the most damaging factors for endothelial integrity, in view of its pro-inflammatory and thrombotic effects12
. In this respect, ecto-AK2 in the SV microvasculature might contribute to the control of local ADP levels via reverse transphosphorylation into ATP and AMP.
In many human and murine cells, AK2 is localized in the mitochondrial intermembrane space, suggesting a role for the protein in providing the energy required for the proliferation of haematopoietic precursors and/or in controlling cell apoptosis. With respect to the latter hypothesis, our findings are reminiscent of the results reported in another type of severe congenital neutropenia caused by HAX-1 deficiency13
. HAX-1 is also located in the mitochondrial intermembrane space and is required to prevent apoptosis of mouse lymphocytes, granulocytes and neurons14,15
. AK2 might also be involved in the regulation of apoptosis via an as yet unknown mechanism which may nevertheless involve its release from the mitochondria, together with cytochrome c16,17
. More recently, AK2 has been implicated in a novel, intrinsic apoptosis pathway, where it forms a complex with FADD and caspase 108
. In this latter report, truncated, mislocated AK2
products were found in the cytosol of human cells and were seen to induce apoptosis. However, our western blot analysis did not reveal the presence of any truncated AK2 protein. This in agreement with the observation of a strong decrease in cell proliferation/survival as well as a block in myeloid differentiation induced by the downregulation of AK2 expression in CD34+ cells. The discrepancy with the published data may be related to the usage of different cell subsets, i.e. HeLa in Lee’s work8
and CD34+ cells in ours. Indeed, no effect on fibroblast cell survival was induced by downregulation of AK2. It is nevertheless possible that formation of an AK2/FADD/caspase 10 complex is required for commitment to the T/NK lymphoid and neutrophil differentiation pathway. Indeed, the recent description of thrombocytopenia in patients carrying mutations in the cytochrome c gene18
emphasizes a possible lineage-specific control of caspase activation that may not be related to the classic cell death mechanism19,20
. Further investigation of the potential involvement of AK2 in regulating apoptosis in certain cell lineages is required.
Taken as a whole, we identified mutations in human AK2 responsible for Reticular Dysgenesis, one of the most severe immunodeficiencies affecting both innate and adaptive immunity and associated with sensorineural deafness. These data enlight the key role of the AK2 protein in haematopoiesis and emphasize a novel cell-lineage restricted pathways controlling energy metabolism and/or cell growth and survival.