In 1964, Zuelzer reported an exceptional congenital neutropenia associated with hyperplasia of mature neutrophils in the bone marrow (BM) – myelokathexis (retention of white blood cells in the BM) 1 . Its acronym (WHIM) derived from the manifestations of Human Papillomavirus (HPV)-induced Warts, Hypogammaglobulinemia, and bacterial Infections together with Myelokathexis 2 . A marked lymphopenia, which affects both T- and B-lymphocytes and NK cells, completes the picture. The clinical onset and complications in WHIM syndrome (WS) are more variable than originally suspected with the notable exceptions of neutropenia and lymphopenia, which are always observed in patients suffering from this disorder 3 . WS is also genetically heterogenous. Most patients present heterozygous autosomal dominant mutations of the gene encoding for CXCR4, the receptor of the CXCL12 chemokine (or Stromal cell Derived Factor-1) 4 , which notably regulates hematopoiesis and peripheral trafficking of neutrophil and lymphocyte subsets. CXCR4 engagement by CXCL12 induces typical activation of Gαi protein-dependent pathways. All CXCR4 mutations described so far result in partial truncations of the receptor’s carboxyl terminal tail (C-tail), with the exception of the recently described missense non truncating E343K mutation 5 , and impair the desensitization process which precludes further G-protein activation thus leading to enhanced and prolonged responsiveness of CXCR4 mutants to CXCL12 (i.e. gain of function) 6 . Leukocytes from the minority of patients who carry a wild-type (WT) CXCR4 gene presented a similar pattern of aberrant CXCL12/CXCR4 responses 4 7 – 9 consistent with a role for these dysfunctions in the WS hematological defects 10 . In support of this assumption, a new knock-in mouse strain that harbors a WS-associated heterozygous mutation of the CXCR4 gene exhibits striking parallels to the major immunological features of WS (i.e. panleukopenia) and is considered as a valuable model of the human syndrome 11 .

An exhaustive literature review since the first description in 1964 identified 52 cases originating from the United States, Japan or Europe (Additional file 1) 1 2 4 5 9 12 – 43 . Recurrent infections may be quite severe, but other presentations are more indolent while the white blood cell count (WBC) appears to be affected in a large range, from mild lympho-neutropenia to near panleukopenia. Therefore, the therapeutic management of these patients is diverse. Some patients have no prophylactic therapy, while others may receive prophylactic antibiotics or antiviral therapies such as Immunoglobulins (Ig), Granulocyte macrophage colony-stimulating factor (GMCSF), Granulocyte colony-stimulating factor (GCSF) and eventually undergo hematopoietic stem cell transplantation 30 . Recently, plerixafor (or AMD3100), a small synthetic antagonist of CXCR4 approved for BM hematopoietic progenitor cells transplantation 44 , has been tested in WS patients and found to promote the mobilization of neutrophils and lymphocytes to the peripheral blood 31 45 . These studies provide the first pharmacological evidence of the causal role of the gain of CXCR4 function in WS-associated panleukopenia and explain the rationale for the use of CXCL12/CXCR4 antagonists in the management of WS. Both the extreme rarity of WS and its clinical heterogeneity prompted us to analyze all cases identified in France through the national Severe Chronic Neutropenia Registry. We collected the patients’ exhaustive clinical, hematological and biological features, as well as their family histories, and performed the genetic and functional analyses of the CXCR4 receptor.

In this study we describe the French national cohort of patients with WS, thereby adding 8 patients and 4 pedigrees to the 52 previously reported distinct cases originating from 32 pedigrees (Additional file 1). The genetic studies performed in 27 pedigrees find that 25 pedigrees (including 3 from this report) bear CXCR4 mutations. R334X, the most frequent mutation, is reported in 15 pedigrees (Table  3). The S338X and S339fs342X mutations are respectively detected in 3 and 2 pedigrees, while the other mutations (E343X, G336X, S341fsX365, the previously unreported G323fs343X mutation herein described and the missense mutation E343K) are reported only once. This survey underlines the extreme rarity of the WS disease, as the birth ratio observed in France can be estimated to 0.23 cases per million births. This incidence rate has been calculated for the period 1990–2006. Nevertheless, we cannot formally exclude the possibility that there may exist some individuals, most likely sporadic cases, with milder clinical courses that do not yet require medical attention and who have therefore not been included in the registry. This low incidence rate, although never calculated worldwide, is in accordance with the scarcity of reported cases as shown in the Additional file 1.

This cohort offers new information about the hematological and infectious profiles of WS. Interestingly, besides the constant neutropenia and lymphopenia, all 8 patients present monocytopenia and half of the patients present profound monocytopenia below 0.1 G/L, contrasting with the monocytosis commonly observed in other congenital neutropenias, such as the elastase neutrophil expressed (ELANE) syndrome. Of particular note, susceptibility to mycobacterial infections may be added to the infection spectrum of WS. Considering the monocytopenia and the infectious profile, composed by pyogenic infections, warts and mycobacteria, WS presents certain similarities with the Mono-MAC syndrome, now identified as the consequence of GATA2 mutations 53 54 . The major phenotypic difference between the two syndromes is the BM myelokathexis feature the WS.

We previously reported, in leukocytes derived from two patients (UPN 5231 and 5446) from pedigree 4 carrying a mutated CXCR4 receptor, that the increased Gαi protein-dependent signaling (e.g. CXCL12-induced chemotaxis) was associated with the inability of CXCR4 to be uncoupled from G proteins (i.e. desensitized) and internalized in response to CXCL12 7 , which is in agreement with other studies (reviewed in 55 56 ). Functional studies could not be assessed in two patients (UPN 5546 and 5449) from pedigree 1 who did not give their consent. In this genetic form of WS, impaired CXCR4 desensitization and internalization resulted from distal truncations of the receptor’s C-tail thereby removing potential phosphorylation sites involved in the attenuation process. Interestingly, we also described a similar pattern of CXCR4 dysfunctions in leukocytes from one subject carrying a WT CXCR4 open reading frame and myelokathexis (UPN 5592) with a full WS phenotype (pedigree 3). This suggests that altered CXCL12/CXCR4-mediated signaling is a common biological trait of WS caused by different genetic mutations, and that for the patients with a WT CXCR4, the genetic cause(s) may involve an effector protein of the CXCL12/CXCR4 axis 7 . Later, fibroblasts and EBV-immortalized B cells derived from this patient were found to display dysfunctions of G protein coupled receptor kinase 3 (GRK3) pointing to the key role of this kinase in the regulation of the CXCR4 receptor attenuation 8 . However, the genetic anomalie(s) causing the GRK3 dysregulation, and the subsequent impairment in CXCR4 inactivation, remain unknown.

Defining WS with certitude is a difficult exercise in the absence of identified CXCR4 mutations since none of the terms of the WS acronym (Warts Hypogammaglobulinemia Infections and Myelokathexis) have full sensibility or specificity. Warts concern 58% of the cases in literature and in agreement with this frequency, five out of eight cases in our cohort. Hypogammaglobulinemia is usually very mild with reported levels between −1 and −2 SD and rarely below. Accordingly, in this cohort, patients presented variable IgG levels with no cases exhibiting frank hypogammaglobulinemia below −3 SD. Although infection susceptibility is on the contrary almost always present, severe infections are rare. Finally, myelokathexis is not pathognomomic of WS, as it is observed in other situations such as in neutropenia linked to the glucose 6 phosphatase, catalytic subunit 3 (G6PC3) 57 , CXCR2 loss-of-function mutations 58 or in gastric cancer 59 . In addition, for three patients from this study, BM smears were initially described as showing only an absence of myeloid blockage, while we identified the typical multi-lobular nuclei of myelokathexis only at the time of the central review. Moreover there is one described WS case with documented CXCR4 mutation but no myelokathexis (P6 41 ). On the contrary, the constant panleukopenia and monocytopenia 3 56 60 are not included in the acronym of the WS. This panleukopenia is also reported in the literature. Indeed, taking into account all original publications displayed in the Additional file 1, the median lowest WBC is 1.65 (mean 1.96; p25: 1 - p75: 2.60 G/L), the median lowest ANC is 0.33 (mean of 0.46; p25: 0.13 - p75: 0.75), with constant lymphopenia and deep monocytopenia (< 0.2 G/l). According to these observations we now suggest WILM (Warts, Infections, Leukopenia, and Myelokathexis) as a new acronym of the syndrome.

To make the definitive diagnosis of WS in the absence of any CXCR4 mutation, we propose that the association of these ‘WILM’ features together with the gain of CXCR4 function are needed. Under such conditions, two pedigrees with WT CXCR4 have been described, one originating from Slovenia 9 15 , and one from our survey, for which a GATA2 mutation was excluded. In addition to these findings, we have studied two patients with GATA2 mutations who presented WS-like features (Mycobacterium avium infection, Warts and Neutropenia) together with an impaired CXCR4 internalization 7 8 61 . This suggests some eventual interplay between CXCR4-dependant signalling and GATA2 that could account for the manifestations of neutropenia and warts of the GATA2 syndrome 62 and beyond, of the more recently described Serine threonine kinase 4 (STK4)-linked syndrome 63 .

Of particular note was the apparent paradox between the profoundly altered WBC and the relatively indolent clinical presentations of WS both in our cohort and in the literature. Indeed, ANC, ALC (including both T- and B-cell subpopulations) and AMC are close to those observed in Severe Combined Immunodeficiency Disease patients. However, the spectrum of infections presented by WS patients is rather limited: they only exhibit a higher susceptibility to bacterial infections from encapsulated Gram-positive and -negative bacteria, staphylococcus and mycobacteria. Among the eleven published cases, for which the bacterial pathogens responsible for infection were identified (Additional file 1), Streptococcus pneumonia was reported in seven cases, Haemophilus influenza in six cases, Staphylococcus aureus in three cases, and Proteus mirabilis, Clostridium perfringens and Pseudomonas aeruginosa in one case each. Finally, apart from two reported cases of herpes infections, one case of severe chicken pox 13 , one case of rubella and two cases of influenza 5 , WS patients mainly present a particular susceptibility to HPV infection that manifests as cutaneous (hand/feet/face) and ano-genital mucosal lesions which abnormally often progress to cancerous lesions. Although we cannot dismiss the hypothesis that the WS immunodeficiency eventually affects anti-HPV immune responses, we rather favor the possibility that the CXCL12/CXCR4 axis represents a host susceptibility factor for HPV-infection and associated carcinogenic evolution 64 . Low risk HPVs that infect the mucosa (such as HPV-6 and −11) do not usually cause cancer in the general population, yet develop as severe dysplasia and carcinoma in WS patients. It must further be pointed out that certain types of infection are not reported in WS such as pulmonar pneumocystosis, zona or severe flu. Thus, the apparent paradox of WS patients is to exhibit a profoundly altered immune function and yet a limited susceptibility to viral and bacterial pathogens, with the notable exception of HPV. This paradox may be explained if we consider that the disease is mainly related to the neutropenia, which, in contrast to other congenital neutropenias, is likely due to an impaired release from the BM that can be transiently overcome during infection, thus resulting in normalization of BM cytology and peripheral neutrophil counts 15 42 43 .

The long-term outcome of WS patients from both our survey and the 52 previously reported patients indicated five deaths (Figure  2A). No death was reported before the age of 20 years with the exception of the medically terminated foetus described in this study. In addition to the two patients herein reported, whose deaths were caused by HPV-induced genital cancer and liver failure, the causes of deaths were lymphoma in two cases (at the ages of 26 28 and 54 years 41 ) and bacterial meningitis in one case (at the age of 31 years 2 ). Three other malignancies were reported including one patient who declared a non-lethal B lymphoma at the age of 31 years followed by a maxillary carcinoma 19 , and another patient who developed a genital cancer of unspecified etiology at age of 20 years 5 . The overall cancer risk in this population is estimated to be about 30% by the age of 40 years, with an age of onset beyond the third decade (Figure  2B). Genital warts, which are reported for 23% of the literature cases and for 28.6% of cases herein described (2 cases), mark a turning point in the natural course of the disease due to their likelihood to progress to intractable multifocal dysplasia and invasive cancer with an early fatal course despite repeated aggressive surgical removal.

Lastly, the association between WS and TOF has been recently reported 65 , which included the patient UPN 5447, reported as P3. This association is strengthened by the herein reported fetus, aborted at 32 gestational weeks due to a double aortic arch, a cardiac abnormality belonging to the same group of conotruncal malformations as TOF.

Even though WS is quite rare, the amount of data collected until now can help to determine the medical management of afflicted patients. The current standard therapeutic approach often associates oral antibiotic prophylaxis, Ig and GCSF. The benefit of Ig prophylaxis is obvious when proposed early in life, as was the case for the two youngest patients of this cohort, but less evident when started later in life, notably after the development of permanent lung damage due to previous infections. The use of GCSF is questionable. Undoubtedly, neutropenia is corrected by GCSF, yet infections do not seem correlated directly to the ANC, and no chronic stomatitis is reported. In addition, patients in our cohort withdrew from GCSF due to the lack of efficiency of the treatment against infections and this pattern was equally frequent in the literature 40 . Long term prophylactic rotational antibiotic therapy is recorded in two patients with COPD (Table  1) in addition to their Ig prophylaxis which seemed to lower the incidence of subsequent pulmonary infection episodes. However, although prophylactic measures can be beneficial on bacterial infections they have no impact on HPV-related disease.

Moreover, for the management of HPV-induced cutaneous and genital warts, standard methods such as cauterization and laser therapy seem relatively inefficient 34 . Aggressive surgical removal, Interferon or Cidofovir administration, as well as topical Imiquimod treatments all proved equally unsatisfying, as lesions tended to reappear. This stresses the need for a better understanding of the physiopathologic mechanisms accounting for the susceptibility to HPV. Prophylactic quadrivalent HPV vaccine administration to a WS patient was recently tested and was associated with protective immunity 25 suggesting that such a vaccine, if its long-term activity can be demonstrated, could be used as early prophylactic treatment for young WS patients and for newly diagnosed WS cases. Hematopoietic stem cell transplantation has been proposed once, but with regards to the natural history of the disease, it seems difficult to propose a pre-emptive program for all patients, as the benefit expected by a transplant is to prevent infections in the third or fourth decade.

We should stress that although the actual management of WS is prophylactic, treatments that specifically target the CXCL12/CXCR4 axis are currently being evaluated. Indeed, two independent phase I clinical trials based on the acute administration of plerixafor have provided the pharmacological evidence of the causal role of CXCR4 dysfunctions in the WS-associated panleukopenia. However, whether such treatment may help to treat or to prevent HPV lesions, EBV lymphoma or Mycobacteria infection, remains to be investigated. Moreover, considering that the management of WS will need efficient and safe in the long run chronic treatments aimed at normalizing, but not abolishing the CXCR4 signaling, and because the long-term safety of AMD3100 may be questioned 66 67 , alternative new inhibitory compounds need to be characterized. The pre-clinical mouse model of the WS 66 67 will be useful for such analyses.

Abs: Antibodies;ALC: Absolute lymphocyte count;AMC: Absolute monocyte count;ANC: Absolute neutrophil count;BM: Bone Marrow;CBC: Complete blood count;COPD: Chronic obstructive pulmonary disease;C-tail: Carboxyl terminal tail;CXCR4: Chemokine (C-X-C motif) receptor type 4;CXCL12: Chemokine (C-X-C motif) ligand 12;ELANE: Elastase neutrophil expressed;EBV: Epstein Barr virus;GCSF: Granulocyte colony-stimulating factor;GMCSF: Granulocyte macrophage colony-stimulating factor;GRK3: G-protein coupled receptor kinase 3;G6PC3: Glucose 6 phosphatase catalytic subunit 3 gene;g.w.: Gestational weeks;Hb: Hemoglobin;HPV: Human Papillomavirus;Ig: Immunoglobulins;PCR: Polymerase chain reaction;SD: Standard deviation;STK4: Serine threonine kinase 4 syndrome;TOF: Tetralogy of Fallot;UPN: Unique patient number;WBC: White blood cell count;WS: WHIM (Warts Hypogammaglobulinemia Infections Myelokathexis) syndrome

The authors declare no competing interests.

OF carried out the bone marrow central review, CBC performed the molecular genetic studies, KB and FB were responsible for the CXCR4 functional studies and analyses. EP, PSR, GD, IP, AD, DK, PB, FF, ALD, GD, JFN, PB, and YB who are referent physicians for the patients, revised the manuscript and contributed to the acquisition and analysis of data with SBC, JD and KB. BB visited each centre yearly to update the register. FB, SBC and JD wrote the manuscript. All authors read and approved the final manuscript.