Chromothripsis is a catastrophic cellular event recently described in cancer in which chromosomes undergo massive deletion and rearrangement. Here, we report a case in which chromothripsis spontaneously cured a patient with WHIM syndrome, an autosomal dominant combined immunodeficiency disease caused by gain-of-function mutation of the chemokine receptor CXCR4. In this patient, deletion of the disease allele, CXCR4 R334X , as well as 163 other genes from one copy of chromosome 2 occurred in a hematopoietic stem cell (HSC) that repopulated the myeloid but not the lymphoid lineage. In competitive mouse bone marrow (BM) transplantation experiments, Cxcr4 haploinsufficiency was sufficient to confer a strong long-term engraftment advantage of donor BM over BM from either wild-type or WHIM syndrome model mice, suggesting a potential mechanism for the patient’s cure. Our findings suggest that partial inactivation of CXCR4 may have general utility as a strategy to promote HSC engraftment in transplantation.

Chromothripsis refers to multiple clustered genetic rearrangements and deletions affecting one or a few chromosomes (). The abnormalities are thought to occur all at once in a single cell, which then presumably either dies or acquires a growth advantage, depending on the genes affected (). Accordingly, chromothripsis was first identified by whole genome sequencing of cancer cell lines and has been reported to affect ∼2% of all cancers (), as well as one patient with a severe congenital cognitive syndrome (). Criteria for chromothripsis include (1) clustering of breakpoints in limited areas of one or several chromosomes with large intervening regions of normal sequence, (2) copy number states that suddenly oscillate between areas of normal heterozygosity and loss of heterozygosity, and (3) rearrangements affecting a single haplotype with multiple fragments rearranged in random orientation and order (). Here, we describe chromothriptic deletions of one copy of chromosome 2, including deletion of the disease allele CXCR4, in a patient with WHIM syndrome that resulted in cure of the disease.

WHIM mutations of CXCR4 increase signaling because they disrupt negative regulatory elements in the carboxy-terminus, thereby exaggerating the normal hematopoietic functions of the receptor (). CXCR4 is normally expressed by most leukocytes and has one ligand, CXCL12 (), which is constitutively expressed at high levels by stromal cells in the bone marrow and normally mediates HSC retention in bone marrow niches (). In addition, CXCR4 signaling promotes hematopoietic stem cell (HSC) quiescence, homing to bone marrow from blood and differentiation into committed myeloid progenitors ().

Transgenic expression of stromal cell-derived factor-1/CXC chemokine ligand 12 enhances myeloid progenitor cell survival/antiapoptosis in vitro in response to growth factor withdrawal and enhances myelopoiesis in vivo.

International Union of Basic and Clinical Pharmacology. LXXXIX. Update on the extended family of chemokine receptors and introducing a new nomenclature for atypical chemokine receptors.

WHIM syndrome is an autosomal dominant combined primary immunodeficiency disease caused by mutations in the chemokine receptor CXCR4 (). The term “WHIM” is an acronym for the main manifestations of the disease: warts, hypogammaglobulinemia, recurrent infections, and myelokathexis; myelokathexis refers to impaired egress of mature neutrophils from bone marrow causing neutropenia (). Most patients with WHIM syndrome are actually panleukopenic, with severely reduced peripheral blood B cells but less severe reductions in peripheral blood T cells and monocytes (). The signature pathogen in WHIM syndrome is human papillomavirus (HPV), which causes warts that cannot be controlled with standard medical treatment and may progress to cancer (). Recurrent bacterial infections also occur, mainly in the sinopulmonary tract, oral cavity, ear, skin, and soft tissue, where chronic complications may arise, especially bronchiectasis and hearing loss. Prophylactic antibiotics, IVIg and G-CSF are often used to reduce the incidence of infections; however, their precise efficacy has not been established (). In contrast, safety and preliminary evidence of clinical efficacy has recently been reported from Phase I studies of the specific CXCR4 antagonist plerixafor (Mozobil, AMD3100) (). Spontaneous remission or cure of WHIM syndrome has not been previously reported.

Description and outcome of a cohort of 8 patients with WHIM syndrome from the French Severe Chronic Neutropenia Registry.

Description and outcome of a cohort of 8 patients with WHIM syndrome from the French Severe Chronic Neutropenia Registry.

Finally, when Cxcr4bone marrow was used in competitive repopulation assays with either Cxcr4or Cxcr4, we found in both cases that the total white blood cell and differential counts in the blood post-transplantation were not significantly altered as compared to reference values for C57Bl/6J mice ( http://phenome.jax.org ), suggesting that Cxcr4genotype does not predispose to myelodysplasia or leukemia in the setting of this transplantation model ( Figure S7 ). Moreover, there is no evidence that Cxcr4mice have a constitutive predisposition to myelodysplasia (data not shown).

Bone marrow cells from Cxcr4 +/o donors on a homozygous CD45.2 background were mixed equally with bone marrow cells from either: Cxcr4 +/S338X donors on a heterozygous CD45.1/CD45.2 background (Cxcr4 +/o /Cxcr4 +/S338X ) or Cxcr4 +/+ donors on a heterozygous CD45.1/CD45.2 background (Cxcr4 +/o /Cxcr4 +/+ ). Five million bone marrow cells from the mixed pairings were then injected intravenously into each CD45.1 recipient mouse that had been lethally irradiated 8 hr before transplantation. Mouse blood was analyzed 39 weeks after bone marrow transplantation for total white blood cell and differential counts (5 recipient mice for each donor pair).

To investigate the mechanism for the apparent competitive advantage of Cxcr4over Cxcr4bone marrow cells for reconstituting the blood, we first measured the proliferative status of the corresponding HSCs in vivo by BrdU incorporation early after transplantation (day 7) ( Figure 6 A). The results indicated ∼20% greater frequency of BrdUCxcr4HSCs as compared to Cxcr4HSCs suggesting a proliferative advantage of Cxcr4HSCs in the bone marrow. To test whether differential retention of leukocytes in the bone marrow might also contribute to the skewed distribution of mature leukocytes in the blood, we sacrificed mice at day 303 after competitive transplantation with Cxcr4and Cxcr4bone marrow cells (the same combination of donor bone marrow cells as were analyzed in the proliferation experiments). The results showed that Cxcr4HSCs, HPCs (hematopoietic progenitor cells), and total CD45cells (mostly mature leukocytes) predominated in the bone marrow by the same 4:1 ratio over the corresponding Cxcr4cells as was found for mature leukocytes in the blood. Thus, the predominance of mature Cxcr4over Cxcr4leukocytes in the blood of competitively transplanted mice cannot be simply explained by low retention of mature Cxcr4leukocytes relative to the retention of mature Cxcr4leukocytes in the bone marrow. Moreover, the results at this very late time point clarify that enhanced Cxcr4HSC proliferation does not result in long-term depletion of HSCs. The frequency distribution of stem and progenitor subtypes was the same for each genotype, indicating no block to progenitor cell differentiation ( Figure 6 B).

(B) Long-term engraftment and differentiation. Bone marrow cells from donor mice with a Cxcr4 +/o genotype on a CD45.2 background were mixed with bone marrow cells from donors with a Cxcr4 +/+ genotype on a CD45.1 background (42:58) and the mixed cells were injected intravenously into lethally irradiated recipient mice. Bone marrow was harvested 303 days later. (i) Gating scheme for long-term HSC (LT-HSC: CD34 − Flt3 − Lin − Sca1 + c-Kit + ), short-term HSC (ST-HSC: CD34 + Flt3 − Lin − Sca1 + c-Kit + ), multipotent progenitors (MPP: CD34 + Flt3 + Lin − Sca1 + c-Kit + ), and common lymphoid progenitors (CLP: IL7ra + Lin − Sca1 low c-Kit low ). (ii) Long-term engraftment. (iii) Differentiation. The distribution frequency of bone marrow cell subsets is similar for Cxcr4 +/o compared to Cxcr4 +/+ donor-derived cell populations. Each data point represents ten mice presented as the mean ± SEM.

(A) Proliferation. Donor bone marrow cells with a Cxcr4 +/o genotype on a homozygous CD45.2 background were mixed with donor bone marrow cells with a Cxcr4 +/+ genotype on a heterozygous CD45.1/CD45.2 background (47:53) and injected intravenously into a lethally irradiated CD45.1 recipient mouse. Six days after bone marrow transplantation, each mouse was given 1.25 mg of BrdU intraperitoneally (i.p.). Twenty hours later, the mice were euthanized for HSC proliferation analysis. (i) Gating scheme for BrdU + HSCs. Bone marrow cells were first gated with CD45.2 (Cxcr4 +/o ) and CD45.1/CD45.2 (Cxcr4 +/+ ), then HSCs were gated as Flt3 − Lin − Sca1 + c-Kit + (Flt3 − LSK), which includes long-term and short-term HSCs, and BrdU + cells were quantitated. (ii) Percentage of BrdU + HSCs in each donor. Data are expressed as mean ± SEM from four mice. The experiment was repeated once with similar results.

To test whether there might be a general selective advantage of Cxcr4 hemizygosity in transplantation, we performed competitive repopulation experiments by mixing ∼2.5 × 10total donor bone marrow cells each from Cxcr4and Cxcr4mice and transplanting the mixture into lethally irradiated Cxcr4recipients. The baseline HSC frequency in bone marrow was the same for Cxcr4and Cxcr4donor mice ( Figure S5 ). In this case, the input ratio was skewed slightly in favor of the Cxcr4cells over the Cxcr4cells (58%:42%). Nevertheless, by day 303 post-transplantation, when the animals were sacrificed, the percentage of Cxcr4neutrophils, monocytes and B cells detectable in the peripheral blood had declined to less than ∼15%, whereas the percentage of each of the corresponding Cxcr4leukocyte subsets had increased to ∼85% with similar kinetics as for the competition with Cxcr4bone marrow ( Figure 5 B). The same effect was observed whether the irradiated recipient mouse was CD45.1, CD45.2, or Cxcr4(data not shown) and whether the donor bone marrows were depleted of lineage-positive cells ( Figure S6 ).

All results shown in (A) and (B) are from a single experiment. Blood draws at weeks 2, 4 and 6 for flow cytometry were performed to determine which mouse bone marrow cell preparations would engraft more readily. Boxes in the FACS plots at the left of each section show the percentage of total donor cells injected that were Lin - . Time course data are shown as the mean ± SEM results from whole bone marrow cell transplanted mice (n = 2) or lineage depleted bone marrow cell transplanted mice (n = 5) for the various blood leukocyte subpopulations shown.

(B) Engraftment advantage of Cxcr4 +/o versus Cxcr4 +/+ lineage-depleted donor bone marrow. Bone marrow cells from donors with a Cxcr4 +/o genotype on a homozygous CD45.2 background and Cxcr4 +/+ on a heterozygous CD45.1/CD45.2 background were mixed equally and then 5 million whole bone marrow cells or 400,000 lineage depleted bone marrow cells were injected intravenously into each CD45.1 recipient mouse that had been lethally irradiated 8 hr before injection.

(A) Engraftment advantage of Cxcr4 +/o versus Cxcr4 +/S338X lineage-depleted donor bone marrow. Bone marrow cells from donors with a Cxcr4 +/o genotype on a homozygous CD45.2 background and Cxcr4 +/S338X on a heterozygous CD45.1/45.2 background were mixed equally and then 5 million whole bone marrow cells (i) or 400,000 lineage depleted bone marrow cells (ii) were injected intravenously into each CD45.1 recipient mouse that had been lethally irradiated 8 hr before injection.

Lineage Depletion of Donor Bone Marrow Cells Does Not Affect the Engraftment Advantage of Cxcr4 Haploinsufficient Cells, Related to Figure 5

To test whether deletion of the disease allele CXCR4alone, and not any of the other 163 genes deleted by chromothripsis would be sufficient to confer a selective advantage to an HSC in the context of WHIM syndrome, we performed competitive repopulation experiments using a mouse model of WHIM syndrome (Cxcr4), hemizygous Cxcr4 mice (Cxcr4, the same as the HSC and myeloid cell CXCR4 genotype of patient WHIM-09), and wild-type (Cxcr4) mice as donors and recipients. Leukocytes from these strains were specifically marked with either CD45.1 or CD45.2 or both, allowing tracking of transplanted donor cell fate in vivo by FACS. When ∼2.5 × 10total donor bone marrow cells from both Cxcr4and Cxcr4mice were mixed together in equal proportions (∼50%:50%) and transplanted into lethally irradiated Cxcr4recipients, by day 105 post-transplantation the percentage of Cxcr4neutrophils, monocytes, and B cells detectable in the peripheral blood had declined to ∼5%, whereas the percentage of each of the corresponding Cxcr4leukocyte subsets had increased to ∼95% ( Figure 5 A). The rate at which the proportion of donor-derived blood cells diverged in frequency from the input ratio of 50:50 occurred in two phases, a rapid early phase within 2 weeks and a slow late phase evident by 2 weeks after transplantation. The inflection point could be earlier than 2 weeks, since this is the first time point when data were collected to allow for recovery after transplantation. Importantly, there was no difference in the baseline HSC frequency in bone marrow for Cxcr4and Cxcr4donor mice, so the number of HSCs transplanted was similar ( Figure S5 ). Since the ANC in the peripheral blood of Cxcr4donor mice is only ∼50% less than the corresponding value in both Cxcr4mice (; J.-L.G., M.S., E.Y., and P.M.M., data not shown ) and Cxcr4mice (J.-L.G., unpublished data), the extreme skewing of these transplantation results in the blood suggested that hemizygous Cxcr4 (Cxcr4) HSCs may have a selective advantage over WHIM (Cxcr4) HSCs for engraftment in this system. Consistent with this, in the bone marrow, as in the blood, we observed a greater content of mature Cxcr4leukocytes compared to mature Cxcr4leukocytes (data not shown). No difference was observed for the frequencies of Cxcr4and Cxcr4T cells in the blood, presumably owing to homeostatic proliferation of mature T cells present in donor bone marrow transferred into irradiated hosts.

(C) Frequency of HSCs. Data are expressed as mean ± SEM from two mice in each group, and repeated once with similar results.

Donor mice used for transplantation experiments were screened for LSK and HSC frequency in bone marrow. The following donor strains were used: Cxcr4 +/o on a CD45.2 congenic background (Cxcr4 +/o (CD45.2)) and its littermates (Cxcr4 +/+ (CD45.2)), and Cxcr4 +/S338X on a CD45.1xCD45.2 background (Cxcr4 +/S338X (CD45.1/CD45.2)) and its littermates (Cxcr4 +/+ (CD45.1/CD45.2)).

Hematopoietic Stem Cell Frequency in Bone Marrow from Donor Mice Is Not Affected by Cxcr4 Haploinsufficiency, Related to Figure 5

Two types of competitive bone marrow transplantation experiments were performed: (A) Cxcr4 +/o versus Cxcr4 +/S338X (mouse model of WHIM syndrome), and (B) Cxcr4 +/o versus Cxcr4 +/+ . (i) Experimental design. (ii) Representative flow cytometry plots demonstrating the relative contributions of CD45 congenic markers in mixed donor bone marrow prior to transplantation (left panel) and in blood after bone marrow transplantation (right panel) in a single mouse. (iii) Cell frequency data for the leukocyte subsets indicated at the top of each panel, presented as the mean ± SEM percentage (%) of total donor-derived cells for each subset (n = 10 mice per data point). SEM was <5% of the mean in all cases and therefore is not visible for most data points. Results were verified in one and two additional independent experiments for (A) and (B), respectively.

Chromothripsis-specific primers did not generate a detectable product by PCR when DNA prepared from the archival spleen sample from patient WHIM-09 was amplified, suggesting that the chromothriptic event occurred after age 9, when splenectomy was performed, but before age 35, when the rise in WBC was already underway ( Figure 4 A). The emergence of uniformly chromothriptic neutrophils, as suggested by WGS, PCR, and bone marrow cytogenetics and FISH, implied that at least some patient HSCs must also be chromothriptic. We verified this with CD34leukocytes cultured from peripheral blood, as well as with CD38CD90CD45RAHSCs FACS-sorted from patient bone marrow ( Figures 4 B, 4C, and S4 ) that both appeared to be markedly deficient in or lack the CXCR4allele by the BstUI PCR-RFLP assay. Consistent with this, CD38CD135CD45RAcommon myeloid precursor cells (CMP) and CD38CD135CD45RAgranulocyte-monocyte precursor cells (GMP) sorted from patient bone marrow also lacked the CXCR4allele by the PCR-RFLP assay, whereas FACS-sorted CD45CD34CD38CD10common lymphoid precursors (CLP) remained CXCR4 Figure 4 D, lower panel). This pattern was confirmed by PCR using chromothripsis-specific primers and DNA from HSCs, CMPs, and CLPs ( Figure 4 D, upper panel). Consistent with this, we found that all mature myeloid cell types tested in both blood and bone marrow lacked or were markedly deficient in the CXCR4WHIM allele, whereas all mature lymphoid cell types tested in peripheral blood and bone marrow were heterozygous CXCR4 Figures 4 B and 4C). This aligns with the cytogenetic results presented in Figure 2 C. Further, we found 100% of EBV-transformed B cell lines (n = 10) prepared from patient blood lacked the chromothriptic chromosome (data not shown). As expected, erythroid precursors, which derive from CMPs, also lacked the CXCR4allele as demonstrated by BstUI PCR-RFLP analysis of Burst Forming Unit-Erythroid (BFU-E) colonies generated from patient CD34cells cultured from PBMCs ex vivo ( Figure 4 E). Thus, the combined evidence suggests that an HSC underwent chromothripsis and selectively repopulated the myeloid lineage (including the erythroid lineage), but not the lymphoid lineage ( Figure 4 F).

Bone marrow aspirate underwent red cell lysis and washing and then was stained with marker specific monoclonal antibodies. An initial sort to enrich CD34 + cells was then performed (left) prior to a second sorting procedure using the indicated gating and sorting strategy (right) to collect HSC and progenitor cells.

Gating Strategy for Purification of Hematopoietic Stem and Progenitor Cells from Bone Marrow of Patient WHIM-09, Related to Figure 4

(F) Summary of myeloid/lymphoid mosaicism for CXCR4 R334X in patient WHIM-09. The immunophenotype used to purify each cell type from enriched CD34 + CD45 + cells is summarized next to each cell type shown. Red, negative for CXCR4 R334X ; green, positive for CXCR4 R334X ; asterisks, purified cell types directly analyzed by PCR-RFLP for the WHIM mutation.

(A–E) Representative results from a BstUI PCR-restriction fragment length polymorphism assay (BstUI), designed to distinguish the wild-type CXCR4 allele (WT) from the CXCR4 R334X WHIM allele (WHIM), as well as from a PCR assay specific for the chromothriptic chromosome (13–16 Jxn). PCR was performed on DNA obtained from the indicated donor leukocyte subsets purified either from blood using magnetic bead purification (B and E) or from a bone marrow aspirate using flow cytometric sorting (C and D). DNA was also prepared from archived WHIM-09 spleen and compared with peripheral blood PMN DNA (A), as well as from Burst-forming Unit-Erythroid colonies and compared with blood leukocyte subsets (E). WHIM-09, index patient; HD, healthy donor; Spl, spleen; PMN, polymorphonuclear leukocytes; 13–16 Jxn, PCR product specific for the chromothriptic junction between segments 13 and 16 of the chromothriptic chromosome of patient WHIM-09; PBMC, peripheral blood mononuclear cells; CD4, purified CD4 + T cells; CD8, purified CD8 + T cells; CD19 purified CD19 + B cells; CD56, purified CD56 + natural killer cells; CD14, purified CD14 + monocytes; CD34, purified CD34 + hematopoietic cells; CD3, purified CD3 + T cells; CD15, purified CD15 + neutrophils; CD16, purified CD16 + neutrophils; HSC, hematopoietic stem cells; CLP, common lymphoid precursor; CMP, common myeloid precursor; GMP, granulocyte/monocyte precursor MEP, megakaryocyte-erythroid precursor; BFU-E, Burst-forming Unit-Erythroid; CXCR4, CXCR4 amplicon not digested with BstUI.

Chromothriptic CXCR4-Haploinsufficient HSC Replacement of the Myeloid Lineage, but Not the Lymphoid Lineage, Is Associated with Clinical Remission in Patient WHIM-09

Figure 4 Chromothriptic CXCR4-Haploinsufficient HSC Replacement of the Myeloid Lineage, but Not the Lymphoid Lineage, Is Associated with Clinical Remission in Patient WHIM-09

The changes seen in the microarray were confirmed by WGS but were actually more complex than had been initially suspected. The derivative chromosome was composed of 18 remaining pieces arranged in random orientation and in a random order, characteristic of chromothripsis ( Figure 3 A). A circular plot of the connections revealed by paired end WGS of the neutrophil DNA is shown in Figure 3 B. The abnormal derivative chromosome, modeled using the breakpoints and connections ( Figure 3 C), revealed a predicted structural banding pattern that was identical to what was observed by cytogenetic analysis. We also developed primer pairs spanning four of the unique chromothriptic boundaries and demonstrated experimentally that they generated the predicted rearranged product from WHIM-09 neutrophil DNA but not from healthy donor DNA (data not shown). By analyzing the rearrangement breakpoints for the creation of novel fusion genes, we found two possibilities, fusions of MFSD2B with LOC285000 and POTEKP with NCKAP5 ( Figure S3 ); however, these genes are unlikely to be expressed or functional because each lacks a promoter and transcription initiation site, and major regions of the potentially fused genes are deleted. Thus, the most likely explanation for the patient’s clinical improvement was haploinsufficiency for one or more of the 164 genes affected by chromothripsis.

(C) Derivative chromothriptic chromosome 2. Ideogram of intact chromosome 2 (left) and a model of the Giemsa cytogenetic banding pattern and the order and orientation of the 18 pieces with the deletions called by microarray shaded in yellow (right) are shown. The resultant remaining chromosome 2 banding pattern predicted by whole genome sequencing closely matches that seen by cytogenetic analysis. Note the location of CXCR4 at 2q22.1 in one of the deleted segments.

(B) Circos plot of chromosome 2 and its normal Giemsa cytogenetic banding pattern labeled from the p arm telomere (0) to the q arm telomere (∼240) in megabases. Large pieces of chromosome 2 were missing from patient WHIM-09 neutrophil DNA and the 18 remaining pieces were arranged in random order. Connections between these pieces and their orientation (inset) are depicted by the colored lines. See Figure S2 and Tables S1 and S2 for additional details. Note that two connections were poorly defined because of the involvement of repetitive centromeric sequence. The inner circular trace is the copy number variation data derived from microarray analysis ( Figures S2 A and S2B). Note that the sites of connections derived from the paired-end sequencing analysis closely match the sites where copy number variation abruptly falls from 2 to 1. The location of CXCR4 is indicated at lower left.

(A) Linear non-proportional plot of the abnormal copy of chromosome 2 in patient WHIM-09 labeled from the p arm telomere (0) to the q arm telomere (243) in megabases with the 18 remaining pieces arranged in their numeric order (top). Connections between these pieces are depicted by the curved lines. Note that some connections were poorly defined because of the involvement of repetitive centromeric sequence (black lines). The order and orientation of the 18 remaining pieces in the derivative chromosome are indicated at the bottom.

Purified neutrophil and cultured skin fibroblast DNA from patient WHIM-09 was isolated and subjected to whole genome sequencing with paired-end analysis.

We next performed whole genome sequencing (WGS) for single nucleotide definition of the event, comparing blood neutrophil and skin fibroblast DNA from WHIM-09 to each other and to the standard human genome sequence in GenBank (version hg19). We obtained an average of 40× coverage of both samples (database of Genotypes and Phenotypes [dbGaP] accession number phs000856.v1.p1. [ http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs000856.v1.p1 ]) and an analysis was performed to locate the DNA reads that had homology with multiple distinct areas of chromosome 2. This technique allowed precise base pair level identification of the inversions and deletions that had occurred on one copy of chromosome 2 ( Table S2 ). No other large deletions or inversions were detected in the genome.

To define the chromosomal abnormalities with greater resolution, microarray was performed using patient bone marrow cell and cultured fibroblast DNA. This revealed that the abnormal chromosome 2 was ∼35 Mb shorter than usual due to seven large deletions ( Figure S2 A), as demonstrated by loss of heterozygosity in these regions and by an abrupt change in the relative copy number from 2 to 1. One of the deletions involved the MYCN gene, confirming the FISH results. In addition, one of the deletions included the position of the CXCR4 gene ( Figure S2 B). This had made the patient hemizygous for CXCR4 in cells having the abnormal chromosome 2 with loss of the CXCR4mutation as well as loss of one copy of 163 other annotated genes ( Table S1 ). Thus, development of hemizygosity of CXCR4, with only the wild-type copy remaining, explained why the CXCR4mutation was not detectable in neutrophil DNA by either DNA sequencing or PCR-RFLP. However, this potentially fortuitous deletion was clearly only a small part of a much larger and more complex genetic event.

(B) Higher resolution microarray view of the 4th and 5th deleted regions reveals that CXCR4 is located in the 5th deleted region (vertical dotted line).

(A) Microarray analysis of bone marrow cell DNA using the Affymetrix Cytoscan HD array. Analysis of copy number state and single nucleotide polymorphism variation using the Chromosome Analysis suite software revealed 7 distinct areas of chromosome 2 that were deleted (red boxes).

The PCR-RFLP assay and DNA sequencing results may both be explained by either reversion or deletion in patient leukocytes of the mutant nucleotide that defines the CXCR4allele. To address this, we first performed cytogenetic analysis of bone marrow cells. In all 20 metaphase cells examined, one copy of chromosome 2, where CXCR4 is located, was acrocentric rather than submetacentric and was significantly shorter than its normal homolog ( Figure 2 A). All other chromosomes appeared normal. In addition to deletions, the banding pattern of the abnormal chromosome 2 suggested the presence of inversions. Fluorescence in situ hybridization (FISH) revealed that the anaplastic lymphoma receptor tyrosine kinase (ALK) gene, at 2p23 on the normal chromosome 2, was on the long arm of the abnormal chromosome 2 ( Figure 2 B). FISH also demonstrated that the abnormal chromosome 2 had a portion of the centromere inverted into the long arm and the N-myc gene (MYCN), normally on 2p24, was absent ( Figure 2 C). Almost all of the interphase bone marrow cells with polymorphic nuclei, consistent with neutrophils, had the abnormal hybridization patterns of the abnormal chromosome 2 ( Figure 2 C), whereas cells with round nuclei had a significantly lower percentage of the abnormal chromosome (data not shown). An immunohistochemical stain of ALK activity on a bone marrow core biopsy section did not reveal abnormal activation of the enzyme despite its abnormal chromosomal location ( Figure S1 ).

Two representative photomicrographs from a WHIM-09 bone marrow biopsy are shown. On the left is a typical hematoxylin and eosin stain and on the right is an immunohistochemical stain for anaplastic lymphoma receptor tyrosine kinase (ALK) activity. Lack of brown color indicates that although the ALK gene is translocated, enzymatic activity is not increased.

(C) FISH with MYCN and CEP 2 probe set (Abbott Molecular). Left: metaphase cell showing the normal chromosome 2 with intact centromere signal (red) and MYCN signal (green) at 2p24. The abnormal chromosome 2 has a portion of its centromere inverted into the long arm splitting the red signal; the green MYCN signal is absent. Right: polymorphonuclear and round interphase nuclei showing the abnormal hybridization pattern with one green MYCN signal and three red CEP 2 signals. One of the red signals is smaller than the other two and is close to one of them. See also Figure S1

(B) FISH with intact ALK break apart probe (Abbott Molecular) signal at 2p23 on normal chromosome 2 and on long arm of abnormal chromosome 2 (der 2).

Massive Deletion and Rearrangement of One Copy of Chromosome 2, the Location of CXCR4, in Patient WHIM-09

Figure 2 Massive Deletion and Rearrangement of One Copy of Chromosome 2, the Location of CXCR4, in Patient WHIM-09

We next genotyped whole blood DNA from family members using an established PCR-BstUI restriction fragment length polymorphism (PCR-RFLP) assay for the most common mutation in WHIM syndrome, CXCR4. Both affected daughters (WHIM-10 and WHIM-11) tested positive, whereas the unaffected husband and third unaffected daughter tested negative. Surprisingly, two independent whole blood samples from WHIM-09, in which the leukocyte content was composed mostly of neutrophils and monocytes ( Figures 1 E and 1F), also tested negative for the mutation. In contrast, DNA from WHIM-09 PBMCs, which were composed mostly of lymphocytes, as well as from a lymphoblastoid cell line generated from WHIM-09 PBMCs both tested positive for CXCR4 Figure 1 E). The mutation was also not detectable by direct sequencing of whole blood cell DNA from WHIM-09, whereas whole blood cell samples from both daughters, WHIM-10 and WHIM-11, were both positive ( Figure 1 F). In contrast, DNA samples from a cheek swab and fibroblasts cultured from a skin biopsy from WHIM-09 were both heterozygous for CXCR4 Figures 1 E and 1G), defining her as a somatic genetic mosaic. A WHIM pedigree with germ-line/somatic genetic mosaicism, where the mother of two affected children was hematologically normal, has previously been described (), but was unlikely to apply to WHIM-09 since she had been markedly symptomatic with severe neutropenia and myelokathexis as a child and cutaneous warts as an adult (). Therefore, we investigated whether a genetic reversion had occurred.

We considered myeloid leukemia or a possible pre-leukemic state as a cause of her mild leukocytosis; however, the patient was clinically well over this ∼20-year time span when her neutrophils and monocytes were increasing, and her blood smear and bone marrow histopathology at the NIH were not consistent with these diagnoses ( Figure 1 D); moreover, specific testing for B and T cell clonality as well as for BCR-ABL and JAK mutations were negative (see Extended Experimental Procedures for details). Consistent with her apparent ∼20-year complete remission of WHIM syndrome by clinical criteria, her bone marrow did not present the characteristic features of the disease (increased myeloid:erythroid ratio, neutrophil vacuolization, eyeglass nuclei in neutrophils), which were, however, all present in her bone marrow histopathology reported in the NEJM in 1964, shown again here for comparison, with permission, in Figure 1 D (). Since the patient reported she had undergone several prior surgeries and blood transfusions, we tested her blood for evidence of allogeneic chimerism and found none (data not shown). Thus, although the patient appeared to be clinically cured, she was hematologically mosaic, with sustained spontaneous correction of neutropenia, monocytopenia, and myelokathexis, and continued deficiency of B and naive T lymphocytes in the blood.

To evaluate potential mechanisms for clinical remission, we first graphed all available white blood cell counts for WHIM-09, including those previously published in the NEJM ( Figure 1 C). Consistent with the clinical history, this revealed severe neutropenia at least from age 9 that was unaffected by splenectomy but that began to resolve spontaneously early in the fourth decade of life, rising slowly over time to a new and stable baseline slightly above the upper limit of normal. The AMC followed the same time course, whereas, interestingly, the ALC did not, starting in the normal range for healthy individuals as a child then increasing inconsistently and only slightly as an adult. Nevertheless, when lymphocyte subsets were examined in detail, all B cell subsets and both naive CD4and CD8T cell subsets were below the lower limit of normal ( Table 1 ), as they were in both daughters and most other patients reported with WHIM syndrome. Consistent with this, WHIM-09 was slightly hypogammaglobulinemic at the time of presentation to NIH with IgG = 535 mg/dl (normal range, 642–1,730 mg/dl). In contrast, memory CD4and CD8T cell subsets were elevated in WHIM-09, but deficient in both daughters. Unfortunately, WHIM-09’s archival lymphocyte subset values from the years when she fulfilled the clinical criteria for WHIM syndrome were not available.

Data are presented as absolute numbers of cells having the indicated immunophenotype per microliter of whole blood.

Based on the values of 11–40 healthy blood donors seen at the NIH Clinical Center.

c Based on the values of 11–40 healthy blood donors seen at the NIH Clinical Center.

Distribution of Leukocyte Subsets in the Peripheral Blood of Index Patient WHIM-09 in Clinical Remission from WHIM Syndrome and Her Two Affected Daughters, WHIM-10 and WHIM-11

Table 1 Distribution of Leukocyte Subsets in the Peripheral Blood of Index Patient WHIM-09 in Clinical Remission from WHIM Syndrome and Her Two Affected Daughters, WHIM-10 and WHIM-11

The index patient, designated WHIM-09, is a white female who presented at age 58 to the NIH requesting evaluation for herself and two of her three daughters, designated WHIM-10 (age 21) and WHIM-11 (age 23) ( Figure 1 A). Both daughters had a history of recurrent infections since early childhood, multiple cutaneous warts, panleukopenia, and hypogammaglobulinemia, and therefore fulfilled all the clinical criteria for WHIM syndrome. In contrast, WHIM-09 reported that from childhood through age 38 she had had many serious infections, often requiring hospitalization, but then none in the 20 subsequent years, and that she had had confluent warts on her hands that spontaneously resolved also in her 30s ( Figure 1 B). Moreover, we found that at the time of presentation, WHIM-09 was not neutropenic, but instead had a mild leukocytosis, including an absolute neutrophil count (ANC) and absolute monocyte count (AMC) that were ∼2-fold greater than the upper limit of normal; in contrast, the absolute lymphocyte count (ALC) was within normal limits. The past medical history revealed that WHIM-09 was in fact the first patient ever described with myelokathexis, the key hematopathologic feature in WHIM syndrome, reported in two articles published in The New England Journal of Medicine (NEJM) in 1964 (). She underwent a therapeutic splenectomy at age 9 for the possibility of autoimmune neutropenia, which was ineffective. There is no evidence that her parents or siblings had WHIM syndrome. Thus, the history and clinical evidence were compatible with a WHIM mutation occurring de novo in patient WHIM-09, autosomal dominant transmission to two of her three daughters, and spontaneous and durable complete clinical remission in WHIM-09 in her fourth decade of life ( Figure 1 A).

(G) Sanger DNA sequencing analysis of DNA from purified peripheral blood neutrophils, cells obtained from a buccal swab (cheek) and cultured skin fibroblasts for patient WHIM-09 in the same region as (F).

(F) Sanger DNA sequencing analysis of whole blood DNA for affected family members in the region near nucleotide position 1,000 (vertical line), the site of WHIM mutation CXCR4 R334X (1,000 C → T). Blue, C; Red, T; Fb, fibroblast; PMN, polymorphonuclear leukocyte (PMN).

(D) Normalization of bone marrow pathology in patient WHIM-09. A representative high magnification (500×) Wright-Giemsa stain of the bone marrow aspirate is shown for the index patient WHIM-09 in 1963 () (reproduced with permission) and in 2013 at ages 9 and 59, respectively. Arrows in left image, eyeglass nuclei in neutrophils.

(C) Spontaneous sustained correction of neutropenia and monocytopenia in patient WHIM-09. WBC, white blood cell count; ANC, absolute neutrophil count; AMC, absolute monocyte count; ALC, absolute lymphocyte count. Arrows indicate age at splenectomy; horizontal lines indicate normal range for each cell type. Note, x axis is discontinuous to show pre/post splenectomy results more clearly.

(B) Spontaneous and complete remission of warts in patient WHIM-09. According to patient WHIM-09, through her fourth decade of life she had had extensive warts on her hands, similar to her daughters (illustrated here for 24-year-old daughter WHIM-11), that spontaneously resolved.

Discussion

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R334X is no longer detectable in key cell types that drive the disease: HSCs, neutrophils, and monocytes. To our knowledge, patient WHIM-09’s spontaneous long-term complete remission of warts without treatment is unprecedented in WHIM syndrome ( Al Ustwani et al., 2014 Al Ustwani O.

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Kurzrock R. A new familial immunodeficiency disorder characterized by severe neutropenia, a defective marrow release mechanism, and hypogammaglobulinemia. We use the word “cure” to refer to the patient’s clinical outcome because she had met all four of the acronymic clinical criteria diagnostic for WHIM syndrome through her fourth decade of life, but has fulfilled none except for mild hypogammaglobulinemia since then (∼20 years, to date). Moreover, the disease allele CXCR4is no longer detectable in key cell types that drive the disease: HSCs, neutrophils, and monocytes. To our knowledge, patient WHIM-09’s spontaneous long-term complete remission of warts without treatment is unprecedented in WHIM syndrome (). Since she remains lymphopenic (B and naive T lymphocytes), this suggests that the myeloid arm of the immune system, probably through monocytes or monocyte-derived cells, plays an essential role in HPV clearance.

The combined evidence suggests that an HSC in WHIM-09 underwent chromothripsis between the second and fourth decade of life and selectively repopulated the myeloid lineage, but not the lymphoid lineage. This pattern of hematologic mosaicism implies that the chromothriptic changes precluded differentiation of HSCs to CLPs but not to CMPs. Thus, the mechanism for maintenance of the B cell lineage, which is WHIM in the patient, is unclear. Possibilities include differentiation from a small population of persistent WHIM HSCs below the level of detection of our assays, or by differentiation of self-sustaining or very long-lived WHIM CLPs. In contrast, the T cell lineage may simply have been maintained by homeostatic proliferation of the pre-chromothriptic WHIM T cell repertoire.

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Jallepalli P.V. Chromothripsis: chromosomes in crisis. R334X in the original chromothriptic HSC of patient WHIM-09, which rendered the cell CXCR4 haploinsufficient (CXCR4+/o), may have been sufficient to repopulate the myeloid lineage—as suggested by the strong HSC engraftment advantage of Cxcr4 haploinsufficient mouse bone marrow cells in our competitive bone marrow transplantation experiments—other factors, particularly haploinsufficiency for one or more of the 163 other genes that were deleted by chromothripsis, may also have contributed. In this regard, at least three of the other 163 deleted genes, DNMT3A ( Challen et al., 2012 Challen G.A.

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Dzierzak E. Interleukin-1 regulates hematopoietic progenitor and stem cells in the midgestation mouse fetal liver. Chromothripsis is a complex chromosomal catastrophe that is thought to occur all at once in one cell (). If the affected cell dies, chromothripsis is clinically silent and undetectable, therefore the true frequency with which it occurs cannot be determined. If the chromothriptic cell acquires a strong selective advantage, it may emerge as a readily detectable, clinically apparent clonal population harboring a single pattern of deletions and rearrangements, resulting for example either in cancer, as previously reported (), or if the location of the event is fortuitous, in cure of a genetic condition, as occurred in patient WHIM-09. Although it is possible that deletion of the disease allele CXCR4in the original chromothriptic HSC of patient WHIM-09, which rendered the cell CXCR4 haploinsufficient (CXCR4), may have been sufficient to repopulate the myeloid lineage—as suggested by the strong HSC engraftment advantage of Cxcr4 haploinsufficient mouse bone marrow cells in our competitive bone marrow transplantation experiments—other factors, particularly haploinsufficiency for one or more of the 163 other genes that were deleted by chromothripsis, may also have contributed. In this regard, at least three of the other 163 deleted genes, DNMT3A (), MYCN (), and IL1R (), have been reported to regulate hematopoiesis ( Table S1 ).

Additional work using more stringent engraftment protocols in mice and ultimately gene editing/transplantation trials in humans will be needed to precisely determine the impact of CXCR4 haploinsufficiency on HSC engraftment as well as on each of the parameters that regulate the physiologic steady-state levels of mature leukocytes and hematopoietic stem and progenitor cells in blood, bone marrow, and other hematopoietic compartments. Such studies may further elucidate precisely how patient WHIM-09 was cured, which may point to general applications in transplantation and gene therapy.