(a) Genealogical trees for 7 families with children suffering from RD (indicated by filled symbols). Diagonal bars indicate deceased individuals. Double horizontal bars indicate consanguineous marriages. AK2 mutations are indicated for each patient and parent.
WT= wild type allele of the AK2 gene. E?= no material available.
(b) Genome-wide linkage analysis Multipoint LOD scores are plotted along the 1p30–p35 region of chromosome 1. CI= confidence interval
(c) Location of AK2 mutations in RD patients. Diagrammatic representation of the AK2A and AK2B encoding regions from exon 1 to 7. The AK2A isoform includes the 6 first exons, while AK2B has an additional exon (VII). The corresponding protein domains are shown in dark grey: NMP binding domain. (NMP), adenylate kinase domain (AK) and LID domain (LID).
(d) Description of the AK2 gene mutations in the 7 RD patients. In 6 patients the mutations were homozygous (**) and in 1 patient heterozygous mutations were found (*).

(a) Western blot analysis of AK2 expression. Lysates from fibroblast (Fib) or B-EBV cell lines derived from RD patients were immunoblotted with an anti-AK2 antibody and with anti-β– tubulin as a loading control.
(b) RT-PCR analysis of AK2 expression. cDNA prepared from fibroblast or B-EBV cell lines derived from RD patients was analyzed by RT-PCR using 5-fold serial dilution. The cDNA input was normalized against 28S-rRNA gene expression.

CD34+ cells isolated from the bone marrow (BM) of patient P3 were transduced with either two bicistronic lentivirus vectors (encoding AK2A and AK2B, respectively, together with a GFP reporter gene: AK2A+B+GFP) or a lentivirus encoding the GFP reporter only (GFP). As a positive control, cord-blood-derived CD34⁺ cells were transduced with GFP. After lentiviral transduction, 10⁴ CD34+ cells from the BM of P3 were seeded in semi-solid medium for each condition, corresponding to 500 complemented cells (based on a transduction efficiency of 5%). As a control, 500 CD34+ cord blood cells were seeded in semi-solid medium (transduction efficiency of 35%).
(a) After 13 days, CFU-G/GM colonies were collected and cytospin preparations of each condition (P3 BM GFP; P3 BM AK2A+B+GFP; CB GFP) were analyzed by May-Grunwald/Giemsa staining. Representative pictures of various stages of neutrophil maturation are shown at magnifications of 50× (left panel) or 100× (right panel).
(b) Enumeration of myeloid subpopulations among CFU-G/GM colonies. Myeloid precursors (blasts, myeloblasts and promyelocytes) and differentiated myeloid cells (myelocytes, metamyelocytes and granulocytes) are represented in percentage and in absolute cell numbers.
(c) Representative flow cytometry analysis of CD15 and CD11b expression in CFU-G/GM colonies (left-hand dot plots) and on transduced GFP+ CFU-G/GM colonies (right-hand dot plots)
(d) Absolute number of double-positive CD15⁺CD11b⁺ labelled cells are indicated for each condition (left panel). The absolute number of GFP⁺ cells isolated from the CFU-G/GM colonies and from the CFU-M colonies are indicated (right panel).

(a) AK2 is not detected in the vestibular epithelium. (b) Top panel. In the post-natal day 7 (PN7) cochlea, AK2 is only detected in the stria vascularis (SV). It is absent from the spiral ligament (SL) and organ of Corti (OC). Lower panel. AK2 is not detected in the SV at birth. (c) In the PN7 SV, AK2 labelling (dark blue) is restricted to the lumen of the capillaries and terminal blood vessels, which are stained green with isolectin. Blood vessels in the spiral ligament (SL) do not contain AK2. Cell nuclei are stained in light blue by Hoechst staining.