IGH 3'RR recombination uncovers a non-germinal center imprint and c-MYC-dependent IGH rearrangement in unmutated chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is an incurable indolent non-Hodgkin lymphoma characterized by tumor B cells that weakly express a B-cell receptor. The mutational status of the variable region (IGHV) within the immunoglobulin heavy chain (IGH) locus is an important prognosis indicator and raises the question of the CLL cell of origin. Mutated IGHV gene CLL are genetically imprinted by activation-induced cytidine deaminase (AID). AID is also required for IGH rearrangements: class switch recombination and recombination between switch Mu (Sµ) and the 3' regulatory region (3'RR) (Sµ-3'RRrec). The great majority of CLL B cells being unswitched led us to examine IGH rearrangement blockade in CLL. Our results separated CLL into two groups on the basis of Sµ-3'RRrec counts per sample: Sµ-3'RRrecHigh cases (mostly unmutated CLL) and Sµ-3'RRrecLow cases (mostly mutated CLL), but not based on the class switch recombination junction counts. Sµ-3'RRrec appeared to be ongoing in Sµ-3'RRrecHigh CLL cells and comparison of Sµ-3'RRrec junction structural features pointed to different B-cell origins for both groups. In accordance with IGHV mutational status and PIM1 mutation rate, Sµ-3'RRrecHigh CLL harbor a non-germinal center experienced B-cell imprint while Sµ-3'RRrecLow CLL are from AID-experienced B cells from a secondary lymphoid organ. In addition to the proposals already made concerning the CLL cell of origin, our study highlights that analysis of IGH recombinatory activity can identify CLL cases from different origins. Finally, on-going Sµ-3'RRrec in Sµ-3'RRrecHigh cells appeared to presumably be the consequence of high c-MYC expression, as c-MYC overexpression potentiated IGH rearrangements and Sµ-3'RRrec, even in the absence of AID for the latter.

Overcoming resistance to αPD-1 of MMR-deficient tumors with high tumor-induced neutrophils levels by combination of αCTLA-4 and αPD-1 blockers.

BACKGROUND: Clinical studies have highlighted the efficacy of anti-programmed death 1 (αPD-1) monoclonal antibodies in patients with DNA mismatch repair-deficient (MMRD) tumors. However, the responsiveness of MMRD cancers to αPD-1 therapy is highly heterogeneous, and the origins of this variability remain not fully understood. METHODS: 4T1 and CT26 mouse tumor cell lines were inactivated for the MMRD gene Msh2, leading to a massive accumulation of mutations after serial passages of cells. Insertions/deletion events and mutation load were evaluated by whole exome sequencing. Mice bearing highly mutated MMRD tumor or parental tumors were treated with αPD-1 and tumor volume was monitored. Immune cell type abundance was dynamically assessed in the tumor microenvironment and the blood by flow cytometry. Neutrophils were depleted in mice using αLY6G antibody, and regulatory T (Treg) cell population was reduced with αCD25 or anti-cytotoxic T-lymphocytes-associated protein 4 (αCTLA-4) antibodies. Patients with MMRD tumors treated with immune checkpoint blockade-based therapy were retrospectively identified and neutrophil-to-lymphocyte ratio (NLR) was evaluated and examined for correlation with clinical benefit. RESULTS: By recapitulating mismatch repair deficiency in different mouse tumor models, we revealed that elevated circulating tumor-induced neutrophils (TIN) in hypermutated MMRD tumors hampered response to αPD-1 monotherapy. Importantly, depletion of TIN using αLy-6G antibody reduced Treg cells and restored αPD-1 response. Conversely, targeting Treg cells by αCD25 or αCTLA-4 antibodies limited peripheral TIN accumulation and elicited response in αPD-1-resistant MMRD tumors, highlighting a crosstalk between TIN and Treg cells. Thus, αPD-1+αCTLA-4 combination overcomes TIN-induced resistance to αPD-1 in mice bearing MMRD tumors. Finally, in a cohort of human (high microsatellite instability)/MMRD tumors we revealed that early on-treatment change in the NLR ratio may predict resistance to αPD-1 therapy. CONCLUSIONS: TIN countered αPD-1 efficacy in MMRD tumors. Since αCTLA-4 could restrict TIN accumulation, αPD-1+αCTLA-4 combination overcomes αPD-1 resistance in hosts with hypermutated MMRD tumors displaying abnormal neutrophil accumulation.

Expression of Constitutive Fusion of Ubiquitin to PCNA Restores the Level of Immunoglobulin A/T Mutations During Somatic Hypermutation in the Ramos Cell Line.

Somatic hypermutation (SHM) of immunoglobulin (Ig) genes is a B cell specific process required for the generation of specific and high affinity antibodies during the maturation of the immune response against foreign antigens. This process depends on the activity of both activation-induced cytidine deaminase (AID) and several DNA repair factors. AID-dependent SHM creates the full spectrum of mutations in Ig variable (V) regions equally distributed at G/C and A/T bases. In most mammalian cells, deamination of deoxycytidine into uracil during S phase induces targeted G/C mutagenesis using either direct replication of uracils or TLS mediated bypass, however only the machinery of activated B lymphocytes can generate A/T mutagenesis around AID-created uracils. The molecular mechanism behind the latter remains incompletely understood to date. However, the lack of a cellular model that reproduces both G/C and A/T mutation spectra constitutes the major hurdle to elucidating it. The few available B cell lines used thus far to study Ig SHM indeed undergo mainly G/C mutations, that make them inappropriate or of limited use. In this report, we show that in the Ramos cell line that undergoes constitutive G/C-biased SHM in culture, the low rate of A/T mutations is due to an imbalance in the ubiquitination/deubiquitination reaction of PCNA, with the deubiquitination reaction being predominant. The inhibition of the deubiquitinase complex USP1-UAF1 or the expression of constitutive fusion of ubiquitin to PCNA provides the missing clue required for DNA polymerase η recruitment and thereafter the introduction of A/T base pair (bp) mutations during the process of IgV gene diversification. This study reports the establishment of the first modified human B cell line that recapitulates the mechanism of SHM of Ig genes in vitro.

TET2 deficiency reprograms the germinal center B cell epigenome and silences genes linked to lymphomagenesis.

The TET2 DNA hydroxymethyltransferase is frequently disrupted by somatic mutations in diffuse large B cell lymphomas (DLBCLs), a tumor that originates from germinal center (GC) B cells. Here, we show that TET2 deficiency leads to DNA hypermethylation of regulatory elements in GC B cells, associated with silencing of the respective genes. This hypermethylation affects the binding of transcription factors including those involved in exit from the GC reaction and involves pathways such as B cell receptor, antigen presentation, CD40, and others. Normal GC B cells manifest a typical hypomethylation signature, which is caused by AID, the enzyme that mediates somatic hypermutation. However, AID-induced demethylation is markedly impaired in TET2-deficient GC B cells, suggesting that AID epigenetic effects are partially dependent on TET2. Last, we find that TET2 mutant DLBCLs also manifest the aberrant TET2-deficient GC DNA methylation signature, suggesting that this epigenetic pattern is maintained during and contributes to lymphomagenesis.

Nfkbie-deficiency leads to increased susceptibility to develop B-cell lymphoproliferative disorders in aged mice.

Aberrant NF-κB activation is a hallmark of most B-cell malignancies. Recurrent inactivating somatic mutations in the NFKBIE gene, which encodes IκBε, an inhibitor of NF-κB-inducible activity, are reported in several B-cell malignancies with highest frequencies in chronic lymphocytic leukemia and primary mediastinal B-cell lymphoma, and account for a fraction of NF-κB pathway activation. The impact of NFKBIE deficiency on B-cell development and function remains, however, largely unknown. Here, we show that Nfkbie-deficient mice exhibit an amplification of marginal zone B cells and an expansion of B1 B-cell subsets. In germinal center (GC)-dependent immune response, Nfkbie deficiency triggers expansion of GC B-cells through increasing cell proliferation in a B-cell autonomous manner. We also show that Nfkbie deficiency results in hyperproliferation of a B1 B-cell subset and leads to increased NF-κB activation in these cells upon Toll-like receptor stimulation. Nfkbie deficiency cooperates with mutant MYD88 signaling and enhances B-cell proliferation in vitro. In aged mice, Nfkbie absence drives the development of an oligoclonal indolent B-cell lymphoproliferative disorders, resembling monoclonal B-cell lymphocytosis. Collectively, these findings shed light on an essential role of IκBε in finely tuning B-cell development and function.

Large deletions in immunoglobulin genes are associated with a sustained absence of DNA Polymerase η.

Somatic hypermutation of immunoglobulin genes is a highly mutagenic process that is B cell-specific and occurs during antigen-driven responses leading to antigen specificity and antibody affinity maturation. Mutations at the Ig locus are initiated by Activation-Induced cytidine Deaminase and are equally distributed at G/C and A/T bases. This requires the establishment of error-prone repair pathways involving the activity of several low fidelity DNA polymerases. In the physiological context, the G/C base pair mutations involve multiple error-prone DNA polymerases, while the generation of mutations at A/T base pairs depends exclusively on the activity of DNA polymerase η. Using two large cohorts of individuals with xeroderma pigmentosum variant (XP-V), we report that the pattern of mutations at Ig genes becomes highly enriched with large deletions. This observation is more striking for patients older than 50 years. We propose that the absence of Pol η allows the recruitment of other DNA polymerases that profoundly affect the Ig genomic landscape.

Whole exome sequencing identifies a new mutation in the SLC19A2 gene leading to thiamine-responsive megaloblastic anemia in an Egyptian family.

BACKGROUND: The Solute Carrier Family 19 Member 2 (SLC19A2, OMIM *603941) encodes the thiamine transporter 1 (THTR-1) that brings thiamine (Vitamin B1) into cells. THTR-1 is the only thiamine transporter expressed in bone marrow, cochlear, and pancreatic beta cells. THTR-1 loss-of-function leads to the rare recessive genetic disease Thiamine-Responsive Megaloblastic Anemia (TRMA, OMIM #249270). METHODS: In vitro stimulated blood lymphocytes were used for cytogenetics and the isolation of genomic DNA used to perform whole exome sequencing (WES). To validate identified mutations, direct Sanger sequencing was performed following PCR amplification. RESULTS: A 6-year-old male born from a consanguineous couple presenting bone marrow failure and microcephaly was referred to our clinic for disease diagnosis. The patient presented a normal karyotype and no chromosomal fragility in response to DNA damage. WES analysis led to the identification of a new pathogenic variant in the SLC19A2 gene (c.596C>G, pSer199Ter) allowing to identify the young boy as a TRMA patient. CONCLUSION: Our analysis extend the number of inactivating mutations in SLC19A2 leading to TRMA that could guide future prenatal diagnosis for the family and follow-up for patients.

A Recurrent Activating Missense Mutation in Waldenström Macroglobulinemia Affects the DNA Binding of the ETS Transcription Factor SPI1 and Enhances Proliferation.

The ETS-domain transcription factors divide into subfamilies based on protein similarities, DNA-binding sequences, and interaction with cofactors. They are regulated by extracellular clues and contribute to cellular processes, including proliferation and transformation. ETS genes are targeted through genomic rearrangements in oncogenesis. The PU.1/SPI1 gene is inactivated by point mutations in human myeloid malignancies. We identified a recurrent somatic mutation (Q226E) in PU.1/SPI1 in Waldenström macroglobulinemia, a B-cell lymphoproliferative disorder. It affects the DNA-binding affinity of the protein and allows the mutant protein to more frequently bind and activate promoter regions with respect to wild-type protein. Mutant SPI1 binding at promoters activates gene sets typically promoted by other ETS factors, resulting in enhanced proliferation and decreased terminal B-cell differentiation in model cell lines and primary samples. In summary, we describe oncogenic subversion of transcription factor function through subtle alteration of DNA binding leading to cellular proliferation and differentiation arrest. SIGNIFICANCE: The demonstration that a somatic point mutation tips the balance of genome-binding pattern provides a mechanistic paradigm for how missense mutations in transcription factor genes may be oncogenic in human tumors.This article is highlighted in the In This Issue feature, p. 681.

TET2 Deficiency Causes Germinal Center Hyperplasia, Impairs Plasma Cell Differentiation, and Promotes B-cell Lymphomagenesis.

TET2 somatic mutations occur in ∼10% of diffuse large B-cell lymphomas (DLBCL) but are of unknown significance. Herein, we show that TET2 is required for the humoral immune response and is a DLBCL tumor suppressor. TET2 loss of function disrupts transit of B cells through germinal centers (GC), causing GC hyperplasia, impaired class switch recombination, blockade of plasma cell differentiation, and a preneoplastic phenotype. TET2 loss was linked to focal loss of enhancer hydroxymethylation and transcriptional repression of genes that mediate GC exit, such as PRDM1. Notably, these enhancers and genes are also repressed in CREBBP-mutant DLBCLs. Accordingly, TET2 mutation in patients yields a CREBBP-mutant gene-expression signature, CREBBP and TET2 mutations are generally mutually exclusive, and hydroxymethylation loss caused by TET2 deficiency impairs enhancer H3K27 acetylation. Hence, TET2 plays a critical role in the GC reaction, and its loss of function results in lymphomagenesis through failure to activate genes linked to GC exit signals. SIGNIFICANCE: We show that TET2 is required for exit of the GC, B-cell differentiation, and is a tumor suppressor for mature B cells. Loss of TET2 phenocopies CREBBP somatic mutation. These results advocate for sequencing TET2 in patients with lymphoma and for the testing of epigenetic therapies to treat these tumors.See related commentary by Shingleton and Dave, p. 1515.This article is highlighted in the In This Issue feature, p. 1494.

B-cell tumor development in Tet2-deficient mice.

The TET2 gene encodes an α-ketoglutarate-dependent dioxygenase able to oxidize 5-methylcytosine into 5-hydroxymethylcytosine, which is a step toward active DNA demethylation. TET2 is frequently mutated in myeloid malignancies but also in B- and T-cell malignancies. TET2 somatic mutations are also identified in healthy elderly individuals with clonal hematopoiesis. Tet2-deficient mouse models showed widespread hematological differentiation abnormalities, including myeloid, T-cell, and B-cell malignancies. We show here that, similar to what is observed with constitutive Tet2-deficient mice, B-cell-specific Tet2 knockout leads to abnormalities in the B1-cell subset and a development of B-cell malignancies after long latency. Aging Tet2-deficient mice accumulate clonal CD19+ B220low immunoglobulin M+ B-cell populations with transplantable ability showing similarities to human chronic lymphocytic leukemia, including CD5 expression and sensitivity to ibrutinib-mediated B-cell receptor (BCR) signaling inhibition. Exome sequencing of Tet2-/- malignant B cells reveals C-to-T and G-to-A mutations that lie within single-stranded DNA-specific activation-induced deaminase (AID)/APOBEC (apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide-like) cytidine deaminases targeted motif, as confirmed by the lack of a B-cell tumor in compound Tet2-Aicda-deficient mice. Finally, we show that Tet2 deficiency accelerates and exacerbates T-cell leukemia/lymphoma 1A-induced leukemogenesis. Together, our data establish that Tet2 deficiency predisposes to mature B-cell malignancies, which development might be attributed in part to AID-mediated accumulating mutations and BCR-mediated signaling.

Hypermutated tumours in the era of immunotherapy: The paradigm of personalised medicine.

Immune checkpoint inhibitors have demonstrated unprecedented clinical activity in a wide range of cancers. Significant therapeutic responses have recently been observed in patients presenting mismatch repair-deficient (MMRD) tumours. MMRD cancers exhibit a remarkably high rate of mutations, which can result in the formation of neoantigens, hypothesised to enhance the antitumour immune response. In addition to MMRD tumours, cancers mutated in the exonuclease domain of the catalytic subunit of the DNA polymerase epsilon (POLE) also exhibit an ultramutated genome and are thus likely to benefit from immunotherapy. In this review, we provide an overview of recent data on hypermutated tumours, including MMRD and POLE-mutated cancers, with a focus on their distinctive clinicopathological and molecular characteristics as well as their immune environment. We also discuss the emergence of immune therapy to treat these hypermutated cancers, and we comment on the recent Food and Drug Administration approval of an immune checkpoint inhibitor, the programmed cell death 1 antibody (pembrolizumab, Keytruda), for the treatment of patients with metastatic MMRD cancers regardless of the tumour type. This breakthrough represents a turning point in the management of these hypermutated tumours and paves the way for broader strategies in immunoprecision medicine.

Diagnosis of Xeroderma pigmentosum variant in a young patient with two novel mutations in the POLH gene.

We describe the characterization of Xeroderma Pigmentosum variant (XPV) in a young Caucasian patient with phototype I, who exhibited a high sensitivity to sunburn and multiple cutaneous tumors at the age of 15 years. Two novel mutations in the POLH gene, which encodes the translesion DNA polymerase η, with loss of function due to two independent exon skippings, are reported to be associated as a compound heterozygous state in the patient. Western blot analysis performed on proteins from dermal fibroblasts derived from the patient and analysis of the mutation spectrum on immunoglobulin genes produced during the somatic hypermutation process in his memory B cells, show the total absence of translesion polymerase η activity in the patient. The total lack of Polη activity, necessary to bypass in an error-free manner UVR-induced pyrimidine dimers following sun exposure, explains the early unusual clinical appearance of this patient.

The IGH locus relocalizes to a "recombination compartment" in the perinucleolar region of differentiating B-lymphocytes.

The immunoglobulin heavy chain (IGH) gene loci are subject to specific recombination events during B-cell differentiation including somatic hypermutation and class switch recombination which mark the end of immunoglobulin gene maturation in germinal centers of secondary lymph nodes. These two events rely on the activity of activation-induced cytidine deaminase (AID) which requires DNA double strand breaks be created, a potential danger to the cell. Applying 3D-fluorescence in situ hybridization coupled with immunofluorescence staining to a previously described experimental system recapitulating normal B-cell differentiation ex vivo, we have kinetically analyzed the radial positioning of the two IGH gene loci as well as their proximity with the nucleolus, heterochromatin and γH2AX foci. Our observations are consistent with the proposal that these IGH gene rearrangements take place in a specific perinucleolar "recombination compartment" where AID could be sequestered thus limiting the extent of its potentially deleterious off-target effects.

V(D)J recombination process and the Pre-B to immature B-cells transition are altered in Fanca-/- mice.

B-lymphocytes in the bone marrow (BM) must generate a functional B-cell receptor and overcome the negative selection induced by reactivity with autoantigens. Two rounds of DNA recombination are required for the production of functional immunoglobulin heavy (Ig-HCs) and light (LCs) chains necessary for the continuation of B-lymphocyte development in the BM. Both rounds depend on the joint action of recombination activating gene-1 (RAG-1) and RAG-2 endonucleases with the DNA non-homologous end-joining pathway. Loss of the FANC gene leads to the chromosome breakage and cancer predisposition syndrome Fanconi anemia. Because the FANC proteins are involved in certain aspects of the recombination process, we sought to determine the impact of the FANC pathway on the Ig diversification process using Fanca-/- mice. In this work we demonstrated that Fanca-/- animals have a mild B-cell differentiation defect characterized by a specific alteration of the IgM- to IgM+ transition of the B220low B-cell population. Pre-B cells from Fanca-/- mice show evidence of impaired kLC rearrangement at the level of the Vk-Jk junction. Furthermore, Fanca-/- mice showed a skewed Vκ gene usage during formation of the LCs Vk-Jk junctions. Therefore, the Fanca protein appears as a yet unidentified factor involved in the primary diversification of Ig.

A single aspartate mutation in the conserved catalytic site of Rev3L generates a hypomorphic phenotype in vivo and in vitro.

Rev3, the catalytic subunit of yeast DNA polymerase ζ, is required for UV resistance and UV-induced mutagenesis, while its mammalian ortholog, REV3L, plays further vital roles in cell proliferation and embryonic development. To assess the contribution of REV3L catalytic activity to its in vivo function, we generated mutant mouse strains in which one or two Ala residues were substituted to the Asp of the invariant catalytic YGDTDS motif. The simultaneous mutation of both Asp (ATA) phenocopies the Rev3l knockout, which proves that the catalytic activity is mandatory for the vital functions of Rev3L, as reported recently. Surprisingly, although the mutation of the first Asp severely impairs the enzymatic activity of other B-family DNA polymerases, the corresponding mutation of Rev3 (ATD) is hypomorphic in yeast and mouse, as it does not affect viability and proliferation and moderately impacts UVC-induced cell death and mutagenesis. Interestingly, Rev3l hypomorphic mutant mice display a distinct, albeit modest, alteration of the immunoglobulin gene mutation spectrum at G-C base pairs, further documenting its role in this process.

129-Derived Mouse Strains Express an Unstable but Catalytically Active DNA Polymerase Iota Variant.

Mice derived from the 129 strain have a nonsense codon mutation in exon 2 of the polymerase iota (Polι) gene and are therefore considered Polι deficient. When we amplified Polι mRNA from 129/SvJ or 129/Ola testes, only a small fraction of the full-length cDNA contained the nonsense mutation; the major fraction corresponded to a variant Polι isoform lacking exon 2. Polι mRNA lacking exon 2 contains an open reading frame, and the corresponding protein was detected using a polyclonal antibody raised against the C terminus of the murine Polι protein. The identity of the corresponding protein was further confirmed by mass spectrometry. Although the variant protein was expressed at only 5 to 10% of the level of wild-type Polι, it retained de novo DNA synthesis activity, the capacity to form replication foci following UV irradiation, and the ability to rescue UV light sensitivity in Polι(-/-) embryonic fibroblasts derived from a new, fully deficient Polι knockout (KO) mouse line. Furthermore, in vivo treatment of 129-derived male mice with Velcade, a drug that inhibits proteasome function, stabilized and restored a substantial amount of the variant Polι in these animals, indicating that its turnover is controlled by the proteasome. An analysis of two xeroderma pigmentosum-variant (XPV) cases corresponding to missense mutants of Polη, a related translesion synthesis (TLS) polymerase in the same family, similarly showed a destabilization of the catalytically active mutant protein by the proteasome. Collectively, these data challenge the prevailing hypothesis that 129-derived strains of mice are completely deficient in Polι activity. The data also document, both for 129-derived mouse strains and for some XPV patients, new cases of genetic defects corresponding to the destabilization of an otherwise functional protein, the phenotype of which is reversible by proteasome inhibition.

Proteomic analysis reveals a FANCA-modulated neddylation pathway involved in CXCR5 membrane targeting and cell mobility.

The aim of this study was to identify novel substrates of the FANCcore complex, the inactivation of which leads to the genetic disorder Fanconi anemia, which is associated with bone marrow failure, developmental abnormalities and a predisposition to cancer. Eight FANC proteins participate in the nuclear FANCcore complex, which functions as an E3 ubiquitin-ligase that monoubiquitylates FANCD2 and FANCI in response to replicative stress. Here, we use mass spectrometry to compare proteins from FANCcore-complex-deficient cells to those of rescued control cells after treatment with hydroxyurea, an inducer of FANCD2 monoubiquitylation. FANCD2 and FANCI appear to be the only targets of the FANCcore complex. We identify other proteins that are post-translationally modified in a FANCA- or FANCC-dependent manner. The majority of these potential targets localize to the cell membrane. Finally, we demonstrate that (a) the chemokine receptor CXCR5 is neddylated; (b) FANCA but not FANCC appears to modulate CXCR5 neddylation through an unknown mechanism; (c) CXCR5 neddylation is involved in targeting the receptor to the cell membrane; and (d) CXCR5 neddylation stimulates cell migration and motility. Our work has uncovered a pathway involving FANCA in neddylation and cell motility.

Fanca deficiency reduces A/T transitions in somatic hypermutation and alters class switch recombination junctions in mouse B cells.

Fanconi anemia is a rare genetic disorder that can lead to bone marrow failure, congenital abnormalities, and increased risk for leukemia and cancer. Cells with loss-of-function mutations in the FANC pathway are characterized by chromosome fragility, altered mutability, and abnormal regulation of the nonhomologous end-joining (NHEJ) pathway. Somatic hypermutation (SHM) and immunoglobulin (Ig) class switch recombination (CSR) enable B cells to produce high-affinity antibodies of various isotypes. Both processes are initiated after the generation of dG:dU mismatches by activation-induced cytidine deaminase. Whereas SHM involves an error-prone repair process that introduces novel point mutations into the Ig gene, the mismatches generated during CSR are processed to create double-stranded breaks (DSBs) in DNA, which are then repaired by the NHEJ pathway. As several lines of evidence suggest a possible role for the FANC pathway in SHM and CSR, we analyzed both processes in B cells derived from Fanca(-/-) mice. Here we show that Fanca is required for the induction of transition mutations at A/T residues during SHM and that despite globally normal CSR function in splenic B cells, Fanca is required during CSR to stabilize duplexes between pairs of short microhomology regions, thereby impeding short-range recombination downstream of DSB formation.

Partial MCM4 deficiency in patients with growth retardation, adrenal insufficiency, and natural killer cell deficiency.

Natural killer (NK) cells are circulating cytotoxic lymphocytes that exert potent and nonredundant antiviral activity and antitumoral activity in the mouse; however, their function in host defense in humans remains unclear. Here, we investigated 6 related patients with autosomal recessive growth retardation, adrenal insufficiency, and a selective NK cell deficiency characterized by a lack of the CD56(dim) NK subset. Using linkage analysis and fine mapping, we identified the disease-causing gene, MCM4, which encodes a component of the MCM2-7 helicase complex required for DNA replication. A splice-site mutation in the patients produced a frameshift, but the mutation was hypomorphic due to the creation of two new translation initiation methionine codons downstream of the premature termination codon. The patients' fibroblasts exhibited genomic instability, which was rescued by expression of WT MCM4. These data indicate that the patients' growth retardation and adrenal insufficiency likely reflect the ubiquitous but heterogeneous impact of the MCM4 mutation in various tissues. In addition, the specific loss of the NK CD56(dim) subset in patients was associated with a lower rate of NK CD56(bright) cell proliferation, and the maturation of NK CD56(bright) cells toward an NK CD56(dim) phenotype was tightly dependent on MCM4-dependent cell division. Thus, partial MCM4 deficiency results in a genetic syndrome of growth retardation with adrenal insufficiency and selective NK deficiency.