Partial loss-of-function mutations in GINS4 lead to NK cell deficiency with neutropenia

Research ArticleCell biologyImmunology Open Access | 10.1172/jci.insight.154948

Matilde I. Conte,1 M. Cecilia Poli,2,3 Angelo Taglialatela,4 Giuseppe Leuzzi,4 Ivan K. Chinn,5,6 Sandra A. Salinas,1,5 Emma Rey-Jurado,2 Nixa Olivares,2 Liz Veramendi-Espinoza,7 Alberto Ciccia,4 James R. Lupski,5,8 Juan Carlos Aldave Becerra,7 Emily M. Mace,1 and Jordan S. Orange1

1Department of Pediatrics, Vagelos College of Physicians and Surgeons, Columbia University Irving Medical Center, New York, New York, USA.

2Faculty of Medicine, Clínica Alemana Universidad del Desarrollo, Santiago, Chile.

3Immunology and Rheumatology Unit, Hospital Roberto del Rio, Santiago, Chile.

4Department of Genetics and Development, Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, New York, USA.

5Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA.

6Division of Immunology, Allergy, and Retrovirology, Texas Children’s Hospital, Houston, Texas, USA.

7Allergy and Clinical Immunology, Hospital Nacional Edgardo Rebagliati Martins, Lima, Peru.

8Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA.

Address correspondence to: Jordan S. Orange, Department of Pediatrics, Columbia University Irving Medical Center, 622 W. 168th St., PH17-201F, New York, New York 10032, USA. Phone: 212.305.2934; Email: jso2121@cumc.columbia.edu.

Authorship note: AT and GL contributed equally to this work.

Find articles by Conte, M. in: JCI | PubMed | Google Scholar

1Department of Pediatrics, Vagelos College of Physicians and Surgeons, Columbia University Irving Medical Center, New York, New York, USA.

2Faculty of Medicine, Clínica Alemana Universidad del Desarrollo, Santiago, Chile.

3Immunology and Rheumatology Unit, Hospital Roberto del Rio, Santiago, Chile.

4Department of Genetics and Development, Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, New York, USA.

5Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA.

6Division of Immunology, Allergy, and Retrovirology, Texas Children’s Hospital, Houston, Texas, USA.

7Allergy and Clinical Immunology, Hospital Nacional Edgardo Rebagliati Martins, Lima, Peru.

8Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA.

Address correspondence to: Jordan S. Orange, Department of Pediatrics, Columbia University Irving Medical Center, 622 W. 168th St., PH17-201F, New York, New York 10032, USA. Phone: 212.305.2934; Email: jso2121@cumc.columbia.edu.

Authorship note: AT and GL contributed equally to this work.

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1Department of Pediatrics, Vagelos College of Physicians and Surgeons, Columbia University Irving Medical Center, New York, New York, USA.

2Faculty of Medicine, Clínica Alemana Universidad del Desarrollo, Santiago, Chile.

3Immunology and Rheumatology Unit, Hospital Roberto del Rio, Santiago, Chile.

4Department of Genetics and Development, Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, New York, USA.

5Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA.

6Division of Immunology, Allergy, and Retrovirology, Texas Children’s Hospital, Houston, Texas, USA.

7Allergy and Clinical Immunology, Hospital Nacional Edgardo Rebagliati Martins, Lima, Peru.

8Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA.

Address correspondence to: Jordan S. Orange, Department of Pediatrics, Columbia University Irving Medical Center, 622 W. 168th St., PH17-201F, New York, New York 10032, USA. Phone: 212.305.2934; Email: jso2121@cumc.columbia.edu.

Authorship note: AT and GL contributed equally to this work.

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1Department of Pediatrics, Vagelos College of Physicians and Surgeons, Columbia University Irving Medical Center, New York, New York, USA.

2Faculty of Medicine, Clínica Alemana Universidad del Desarrollo, Santiago, Chile.

3Immunology and Rheumatology Unit, Hospital Roberto del Rio, Santiago, Chile.

4Department of Genetics and Development, Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, New York, USA.

5Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA.

6Division of Immunology, Allergy, and Retrovirology, Texas Children’s Hospital, Houston, Texas, USA.

7Allergy and Clinical Immunology, Hospital Nacional Edgardo Rebagliati Martins, Lima, Peru.

8Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA.

Address correspondence to: Jordan S. Orange, Department of Pediatrics, Columbia University Irving Medical Center, 622 W. 168th St., PH17-201F, New York, New York 10032, USA. Phone: 212.305.2934; Email: jso2121@cumc.columbia.edu.

Authorship note: AT and GL contributed equally to this work.

Find articles by Leuzzi, G. in: JCI | PubMed | Google Scholar |

1Department of Pediatrics, Vagelos College of Physicians and Surgeons, Columbia University Irving Medical Center, New York, New York, USA.

2Faculty of Medicine, Clínica Alemana Universidad del Desarrollo, Santiago, Chile.

3Immunology and Rheumatology Unit, Hospital Roberto del Rio, Santiago, Chile.

4Department of Genetics and Development, Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, New York, USA.

5Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA.

6Division of Immunology, Allergy, and Retrovirology, Texas Children’s Hospital, Houston, Texas, USA.

7Allergy and Clinical Immunology, Hospital Nacional Edgardo Rebagliati Martins, Lima, Peru.

8Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA.

Address correspondence to: Jordan S. Orange, Department of Pediatrics, Columbia University Irving Medical Center, 622 W. 168th St., PH17-201F, New York, New York 10032, USA. Phone: 212.305.2934; Email: jso2121@cumc.columbia.edu.

Authorship note: AT and GL contributed equally to this work.

Find articles by Chinn, I. in: JCI | PubMed | Google Scholar |

1Department of Pediatrics, Vagelos College of Physicians and Surgeons, Columbia University Irving Medical Center, New York, New York, USA.

2Faculty of Medicine, Clínica Alemana Universidad del Desarrollo, Santiago, Chile.

3Immunology and Rheumatology Unit, Hospital Roberto del Rio, Santiago, Chile.

4Department of Genetics and Development, Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, New York, USA.

5Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA.

6Division of Immunology, Allergy, and Retrovirology, Texas Children’s Hospital, Houston, Texas, USA.

7Allergy and Clinical Immunology, Hospital Nacional Edgardo Rebagliati Martins, Lima, Peru.

8Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA.

Address correspondence to: Jordan S. Orange, Department of Pediatrics, Columbia University Irving Medical Center, 622 W. 168th St., PH17-201F, New York, New York 10032, USA. Phone: 212.305.2934; Email: jso2121@cumc.columbia.edu.

Authorship note: AT and GL contributed equally to this work.

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1Department of Pediatrics, Vagelos College of Physicians and Surgeons, Columbia University Irving Medical Center, New York, New York, USA.

2Faculty of Medicine, Clínica Alemana Universidad del Desarrollo, Santiago, Chile.

3Immunology and Rheumatology Unit, Hospital Roberto del Rio, Santiago, Chile.

4Department of Genetics and Development, Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, New York, USA.

5Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA.

6Division of Immunology, Allergy, and Retrovirology, Texas Children’s Hospital, Houston, Texas, USA.

7Allergy and Clinical Immunology, Hospital Nacional Edgardo Rebagliati Martins, Lima, Peru.

8Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA.

Address correspondence to: Jordan S. Orange, Department of Pediatrics, Columbia University Irving Medical Center, 622 W. 168th St., PH17-201F, New York, New York 10032, USA. Phone: 212.305.2934; Email: jso2121@cumc.columbia.edu.

Authorship note: AT and GL contributed equally to this work.

Find articles by Rey-Jurado, E. in: JCI | PubMed | Google Scholar

1Department of Pediatrics, Vagelos College of Physicians and Surgeons, Columbia University Irving Medical Center, New York, New York, USA.

2Faculty of Medicine, Clínica Alemana Universidad del Desarrollo, Santiago, Chile.

3Immunology and Rheumatology Unit, Hospital Roberto del Rio, Santiago, Chile.

4Department of Genetics and Development, Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, New York, USA.

5Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA.

6Division of Immunology, Allergy, and Retrovirology, Texas Children’s Hospital, Houston, Texas, USA.

7Allergy and Clinical Immunology, Hospital Nacional Edgardo Rebagliati Martins, Lima, Peru.

8Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA.

Address correspondence to: Jordan S. Orange, Department of Pediatrics, Columbia University Irving Medical Center, 622 W. 168th St., PH17-201F, New York, New York 10032, USA. Phone: 212.305.2934; Email: jso2121@cumc.columbia.edu.

Authorship note: AT and GL contributed equally to this work.

Find articles by Olivares, N. in: JCI | PubMed | Google Scholar

1Department of Pediatrics, Vagelos College of Physicians and Surgeons, Columbia University Irving Medical Center, New York, New York, USA.

2Faculty of Medicine, Clínica Alemana Universidad del Desarrollo, Santiago, Chile.

3Immunology and Rheumatology Unit, Hospital Roberto del Rio, Santiago, Chile.

4Department of Genetics and Development, Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, New York, USA.

5Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA.

6Division of Immunology, Allergy, and Retrovirology, Texas Children’s Hospital, Houston, Texas, USA.

7Allergy and Clinical Immunology, Hospital Nacional Edgardo Rebagliati Martins, Lima, Peru.

8Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA.

Address correspondence to: Jordan S. Orange, Department of Pediatrics, Columbia University Irving Medical Center, 622 W. 168th St., PH17-201F, New York, New York 10032, USA. Phone: 212.305.2934; Email: jso2121@cumc.columbia.edu.

Authorship note: AT and GL contributed equally to this work.

Find articles by Veramendi-Espinoza, L. in: JCI | PubMed | Google Scholar

1Department of Pediatrics, Vagelos College of Physicians and Surgeons, Columbia University Irving Medical Center, New York, New York, USA.

2Faculty of Medicine, Clínica Alemana Universidad del Desarrollo, Santiago, Chile.

3Immunology and Rheumatology Unit, Hospital Roberto del Rio, Santiago, Chile.

4Department of Genetics and Development, Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, New York, USA.

5Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA.

6Division of Immunology, Allergy, and Retrovirology, Texas Children’s Hospital, Houston, Texas, USA.

7Allergy and Clinical Immunology, Hospital Nacional Edgardo Rebagliati Martins, Lima, Peru.

8Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA.

Address correspondence to: Jordan S. Orange, Department of Pediatrics, Columbia University Irving Medical Center, 622 W. 168th St., PH17-201F, New York, New York 10032, USA. Phone: 212.305.2934; Email: jso2121@cumc.columbia.edu.

Authorship note: AT and GL contributed equally to this work.

Find articles by Ciccia, A. in: JCI | PubMed | Google Scholar

1Department of Pediatrics, Vagelos College of Physicians and Surgeons, Columbia University Irving Medical Center, New York, New York, USA.

2Faculty of Medicine, Clínica Alemana Universidad del Desarrollo, Santiago, Chile.

3Immunology and Rheumatology Unit, Hospital Roberto del Rio, Santiago, Chile.

4Department of Genetics and Development, Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, New York, USA.

5Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA.

6Division of Immunology, Allergy, and Retrovirology, Texas Children’s Hospital, Houston, Texas, USA.

7Allergy and Clinical Immunology, Hospital Nacional Edgardo Rebagliati Martins, Lima, Peru.

8Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA.

Address correspondence to: Jordan S. Orange, Department of Pediatrics, Columbia University Irving Medical Center, 622 W. 168th St., PH17-201F, New York, New York 10032, USA. Phone: 212.305.2934; Email: jso2121@cumc.columbia.edu.

Authorship note: AT and GL contributed equally to this work.

Find articles by Lupski, J. in: JCI | PubMed | Google Scholar

1Department of Pediatrics, Vagelos College of Physicians and Surgeons, Columbia University Irving Medical Center, New York, New York, USA.

2Faculty of Medicine, Clínica Alemana Universidad del Desarrollo, Santiago, Chile.

3Immunology and Rheumatology Unit, Hospital Roberto del Rio, Santiago, Chile.

4Department of Genetics and Development, Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, New York, USA.

5Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA.

6Division of Immunology, Allergy, and Retrovirology, Texas Children’s Hospital, Houston, Texas, USA.

7Allergy and Clinical Immunology, Hospital Nacional Edgardo Rebagliati Martins, Lima, Peru.

8Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA.

Address correspondence to: Jordan S. Orange, Department of Pediatrics, Columbia University Irving Medical Center, 622 W. 168th St., PH17-201F, New York, New York 10032, USA. Phone: 212.305.2934; Email: jso2121@cumc.columbia.edu.

Authorship note: AT and GL contributed equally to this work.

Find articles by Aldave Becerra, J. in: JCI | PubMed | Google Scholar

1Department of Pediatrics, Vagelos College of Physicians and Surgeons, Columbia University Irving Medical Center, New York, New York, USA.

2Faculty of Medicine, Clínica Alemana Universidad del Desarrollo, Santiago, Chile.

3Immunology and Rheumatology Unit, Hospital Roberto del Rio, Santiago, Chile.

4Department of Genetics and Development, Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, New York, USA.

5Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA.

6Division of Immunology, Allergy, and Retrovirology, Texas Children’s Hospital, Houston, Texas, USA.

7Allergy and Clinical Immunology, Hospital Nacional Edgardo Rebagliati Martins, Lima, Peru.

8Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA.

Address correspondence to: Jordan S. Orange, Department of Pediatrics, Columbia University Irving Medical Center, 622 W. 168th St., PH17-201F, New York, New York 10032, USA. Phone: 212.305.2934; Email: jso2121@cumc.columbia.edu.

Authorship note: AT and GL contributed equally to this work.

Find articles by Mace, E. in: JCI | PubMed | Google Scholar |

1Department of Pediatrics, Vagelos College of Physicians and Surgeons, Columbia University Irving Medical Center, New York, New York, USA.

2Faculty of Medicine, Clínica Alemana Universidad del Desarrollo, Santiago, Chile.

3Immunology and Rheumatology Unit, Hospital Roberto del Rio, Santiago, Chile.

4Department of Genetics and Development, Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, New York, USA.

5Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA.

6Division of Immunology, Allergy, and Retrovirology, Texas Children’s Hospital, Houston, Texas, USA.

7Allergy and Clinical Immunology, Hospital Nacional Edgardo Rebagliati Martins, Lima, Peru.

8Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA.

Address correspondence to: Jordan S. Orange, Department of Pediatrics, Columbia University Irving Medical Center, 622 W. 168th St., PH17-201F, New York, New York 10032, USA. Phone: 212.305.2934; Email: jso2121@cumc.columbia.edu.

Authorship note: AT and GL contributed equally to this work.

Find articles by Orange, J. in: JCI | PubMed | Google Scholar |

Authorship note: AT and GL contributed equally to this work.

Published November 8, 2022 - More info

Published in Volume 7, Issue 21 on November 8, 2022
JCI Insight. 2022;7(21):e154948. https://doi.org/10.1172/jci.insight.154948.
© 2022 Conte et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published November 8, 2022 - Version history
Received: September 14, 2021; Accepted: September 14, 2022 View PDF Abstract

Human NK cell deficiency (NKD) is a primary immunodeficiency in which the main clinically relevant immunological defect involves missing or dysfunctional NK cells. Here, we describe a familial NKD case in which 2 siblings had a substantive NKD and neutropenia in the absence of other immune system abnormalities. Exome sequencing identified compound heterozygous variants in Go-Ichi-Ni-San (GINS) complex subunit 4 (GINS4, also known as SLD5), an essential component of the human replicative helicase, which we demonstrate to have a damaging impact upon the expression and assembly of the GINS complex. Cells derived from affected individuals and a GINS4-knockdown cell line demonstrate delayed cell cycle progression, without signs of improper DNA synthesis or increased replication stress. By modeling partial GINS4 depletion in differentiating NK cells in vitro, we demonstrate the causal relationship between the genotype and the NK cell phenotype, as well as a cell-intrinsic defect in NK cell development. Thus, biallelic partial loss-of-function mutations in GINS4 define a potentially novel disease-causing gene underlying NKD with neutropenia. Together with the previously described mutations in other helicase genes causing NKD, and with the mild defects observed in other human cells, these variants underscore the importance of this pathway in NK cell biology.

Graphical Abstractgraphical abstract Introduction

NK cells are lymphocytes of the innate immune system that critically function in targeting virally infected and tumorigenic cells. They exert rapid cytotoxic functions by forming synaptic contacts with susceptible target cells and secreting effector molecules, including granzymes and perforin contained in preformed lytic granules, at the lytic immune synapse (1). As components of the innate immune system, NK cells express germline-encoded activating and inhibitory receptors whose balance modulates their capability to either spare healthy or kill diseased cells (2).

In humans, NK cells are typically identified as CD3− and CD56+ lymphocytes comprising 5%–20% of those in peripheral blood, and they populate primary and secondary lymphoid organs, as well as peripheral tissues (3). NK cells are highly heterogeneous, although 2 major subsets in peripheral blood are classically described as CD3−CD56brightCD16− and CD3−CD56dimCD16+, each having different functional properties. CD56bright cells potently produce cytokines and are more frequently found in tissues, whereas CD56dim cells excel in cytotoxic functions and represent approximately 90% of circulating NK cells (4). NK cells originate from hematopoietic stem cells (HSC) in BM and undergo terminal maturation in secondary lymphoid tissues. CD56bright cells can be direct precursors of CD56dim NK cells as demonstrated by their: (a) having longer telomeres (5); (b) appearing more rapidly after BM transplantation (6); (c) being able to transition in vitro to CD56dim (7); and (d) developing into CD16+ NK cells in humanized immune mice (8). Only BM is essential for the generation of NK cells, but their development is shaped by the secondary lymphoid tissue microenvironment (9).

NK cell deficiency (NKD) is a primary immunodeficiency in which NK cells are nonfunctional, missing, or have dysregulated terminal maturation (10) and in which NK cell abnormalities account for the majority of the clinically relevant immunological defects. NKD has the striking clinical hallmark of unusual susceptibility to severe and atypical manifestations of herpesviruses, including cytomegalovirus (CMV), varicella zoster virus (VZV), herpes simplex virus (HSV), EBV, and/or problems with papilloma viruses, highlighting the importance of NK cells for host defense (1012). The study of patients with NKD provides a meaningful opportunity to understand the complex process and requirements of human NK cell development and function.

Currently, 7 NKD-causing monogenic conditions have been described due to pathogenic variants in the following genes: GATA binding domain protein 2 (GATA2), IFN regulatory factor 8 (IRF8), Fc fragment of IgG receptor IIIa (FCGR3A), regulator of telomere elongation helicase I (RTEL1), minichromosome maintenance complex component 4 (MCM4), Go-Ichi-Ni-San (GINS) complex subunit 1 (GINS1), and minichromosome maintenance 10 replication initiation factor (MCM10) (1321). NKD caused by FCGR3A variants is referred to as a “functional NKD,” as only NK cell function is impaired, with normal NK cell maturation and development. The other 6 described NKD conditions can be labeled “classical NKD” and are defined by low or absent NK cell numbers or abnormal distribution of NK cell subsets, consistent with impaired NK cell development or maturation.

Of note, 3 of the 6 classical NKDs are caused by impactful variants in genes that comprise the replicative DNA helicase, underscoring the importance of this complex and, more broadly, the regulation of DNA replication and the cell cycle in human NK cell development. MCM4 deficiency leads to reduced NK cells in the periphery, with a specific reduction of the CD56dim NK cell subset and concomitant overrepresentation of the CD56bright subset (15). While NKD is the major immunological manifestation of human MCM4 deficiency, the clinical phenotype of affected individuals also includes short stature, adrenal insufficiency, and microcephaly (15, 17, 22). Similarly, rare and damaging biallelic variants in GINS1 cause short stature, dysmorphic features, and decreased NK cell frequencies with an increased CD56bright/CD56dim ratio. GINS1 cases represent the originally described familial NKD and, additionally, have chronic neutropenia that can be corrected with the administration of G-CSF (14, 23, 24). As predicted by their cellular function, partial loss-of-function (LoF) mutations in MCM4 and GINS1 result in impaired DNA replication with increased DNA damage. In the case of MCM4 deficiency, increased apoptosis in the CD56bright NK cell subset was reversible by treatment with IL-2 (15). However, these CD56bright NK cells did not proliferate at the same rate as unaffected control cells in response to stimulation with IL-2. This finding suggested that the accumulation of chromosomal aberrations during proliferation of the CD56bright subset leads to the generation of only a few CD56dim NK cells, strengthening the preexisting hypothesis that CD56dim NK cells arise from the CD56bright subset.

Recently, NKD was reported due to biallelic variants in MCM10 and characterized by a near absence of NK cells in the proband with increased frequency of CD56bright NK cells (19). These variants cause replication stress (RS), increased genomic instability, and telomere maintenance defects in multiple cell types; however, the predominant immune defect consisted of NKD (19, 25). Recapitulation of NK cell development using MCM10-knockdown (MCM10-KD) CD34+ cells, and patient-derived iPSCs in a humanized mouse model, demonstrated that loss of MCM10 function leads to impaired NK cell maturation and decreased survival of NK cells, further pointing to the reliance of NK cell development upon the replicative DNA helicase (19).

The human CMG helicase (CDC45, MCM2-7, and GINS1-4) complex is essential for dsDNA unwinding at the replication fork. The MCM2-7 ring is the core helicase component and is loaded as an inactive double hexamer at thousands of replication origins during the G1 phase in a process called “origin licensing” (26). The activation of MCM2-7 occurs during the early S phase and is tightly regulated by recruiting the firing factors CDC45 and the GINS1-4 tetramer. For proper initiation and elongation of DNA synthesis, the association of MCM10, CTF4, and subsequent DNA polymerases to the CMG is required (27). Inactive MCM2-7 complexes are usually expressed in excess compared with the number of replication forks formed in S phase and are distributed at locations distant from the origins, presenting a paradox called the “MCM paradox” (28). In addition to supplying backup origins under RS, inactive origins serve to function in fork speed management in order to prevent genome instability and tumor formation (29). The mechanism by which biallelic partial LoF mutations in replicative helicase genes uniquely leads to NK cell abnormalities — and neutropenia, in the case of GINS1 deficiency — while sparing most other immune cells is poorly understood. Here, we describe a case of biallelic loss of another CMG helicase component, GINS4, resulting in NKD with neutropenia. By cellular modeling of the GINS4 variants, we validate the damaging impact of both variants on GINS complex expression and stability and establish a requirement for GINS4 in NK cell development.

Results

Clinical presentation and immunological phenotype. Two siblings, born to nonconsanguineous healthy parents, presented with suspected immunodeficiency. The proband (II.1) had intrauterine growth restriction, growth delay, tonsillar hypertrophy, cryptorchidism, and localized BCG infection after vaccination. At 11 months of age, he developed generalized seizures with intracranial calcifications, suggesting a history of CMV infection; however, outside of elevated CMV-IgG, additional clinical studies were not performed at that time. Subsequently, within the first 3 years of life, he developed pneumonia, sinusitis, intermittent diarrhea, gastrointestinal sepsis, oral abscess, gingivitis, recurrent otitis, and severe varicella infection with a necrotizing nasolabial ulcer. Currently, he continues to have intermittent diarrhea and recurrent herpes labialis. The proband’s younger sister (II.2) had experienced non–life-threatening varicella at 10 months of age and has recurrent herpes labialis (Table 1).

Individuals II.1 and II.2 had normal T and B cell counts, low NK cell counts, normal or increased immunoglobulin levels, and neutropenia (more substantive in individual II.1) (Supplemental Table 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.154948DS1). BM analysis in the proband showed incomplete neutrophil maturation. He was successfully treated with G-CSF starting at 3 years of age, initially daily and currently twice a week, resulting in some clinical improvement of gingivitis, fever, and diarrhea. His sister was treated monthly with G-CSF starting at 7 months of age and for 2 years. She is no longer receiving any treatment and has stable moderate neutrophil counts. The proband’s parents were largely unaffected, although the mother has recurrent herpes labialis. The proband’s maternal uncle died from severe gastrointestinal sepsis at 9 months of age.

Given the viral infections and low NK cell counts, research-level evaluations were performed. Flow cytometry analysis demonstrated consistent NK cell abnormalities in both individuals II.1 and II.2. The frequency of CD56+CD3− NK cells was substantially decreased, with values ranging from 0.002% to 1.34 % in individual II.1 and from 0.1% to 3.01% in individual II.2, with an increased proportion of the CD56bright NK subset and decreased CD16+ NK cells (Figure 1A and Table 2). We further evaluated the expression of the NK cell maturation markers CD57 and CD94. Lower MFI for both was observed at baseline in both siblings’ NK cells, as well in their CD56dim and CD56bright subsets. Interestingly, the expression of CD94 and CD57 did not increase on NK cells after stimulation with IL-15 for 48 hours (Supplemental Figure 1A).

Frequency and cytotoxic function of peripheral NK cells.Figure 1

Frequency and cytotoxic function of peripheral NK cells. (A) Flow cytometric analysis of CD56+CD3− NK cells and CD56+CD3+ NKT (top) and frequency of CD16+ NK cells (bottom). Two healthy donors, siblings (II.1, II.2), and their parents (I.1, I.2) are shown. Data are from a representative experiment of 3 independent repeats summarized in Table 2. LC, laboratory control; SC, shipping control. (B) 51Cr release assay to evaluate the cytotoxic function against K562 is shown for siblings, healthy parents, and LC. Data are shown as mean ± SD of technical replicates. Data are from a representative experiment of 2 independent repeats. (C) Graph of the lytic units calculated from the values of the 51Cr release assay shown in B.

We also evaluated NK cells for the expression of the effector molecules perforin and IFN-γ and CD107a as an indicator of activation and degranulation upon stimulation of PBMC with phorbol 12-myristate 13-acetate ionomycin (PMA-iono). The levels of CD107a and perforin expression per NK cell were seemingly normal in both individuals. The IFN-γ production, however, was lower in II.2 than in the HD, despite an increased frequency of NK cells (3%) compared with previous evaluations (Supplemental Figure 1B). Notably, survival of the proband’s NK cells upon stimulation with PMA was reduced.

To evaluate NK cell cytotoxic function, chromium 51 (51Cr) release assays were performed for all family members and were compared with that of healthy donors (HDs). Very low lytic activity against K562 target cells was identified in II.1 and II.2 (Figure 1B), which was not rescued by adding IL-2 to the assay (data not shown). Their mother (I.1) also had reduced NK cell cytotoxic function, despite seemingly normal frequencies of NK cells. The cytotoxic activity was still reduced when expressed as lytic unit (LU) per PBMCs (Figure 1C, left panel). This difference was no longer present in individual II.2 when we normalized for the extreme underrepresentation of NK cells to define the LU per NK cells; thus, the impaired cytotoxic activity is likely attributed to the lower frequency of NK cells in this individual. The normalized analysis, however, identifies lower LU per NK cells in individual I.1, the proband’s mother (Figure 1C, right panel).

The limited availability of primary samples prevented extensive immunological evaluation; nevertheless, we investigated some circulating T cell subsets. Individuals II.1 and II.2 had normal numbers and percentages of naive and memory CD4+ and CD8+ T cells (Supplemental Figure 2A). Also, mucosal-associated invariant T cells (MAIT) and γδ-T subsets were seemingly normal (Supplemental Figure 2B).

In aggregate, the clinical history of the proband in light of the NK cell phenotypic and functional data suggested the familial presence of immunodeficiency with a major defect in NK cell numbers and resulting lytic function.

Compound heterozygous variants in GINS4 identified via exome sequencing. Exome sequencing on DNA from blood samples of the proband, his sister, and their parents was performed. Analyses were executed in accordance with dominant and recessive models of Mendelian inheritance. Given that both parents were clinically unaffected, we hypothesized a recessive model of inheritance. Poor quality and frequent variants (minor allelic frequency > 0.001) were filtered out, and focus was given to biallelic variants. The proband and his sister shared 2 heterozygous compound variants in exon 7 of GINS4, each inherited from an individual parent. These changes encompassed a missense variant encoding a valine-to-leucine change, NM_032336:exon7:c.511G>C:p.V171L, and a stop gain variant, NM_032336:exon7:c.C571T:p.Q191X (Figure 2A), both mapping to the C-terminal domain B of GINS4 (Figure 2, B and C). Segregation of both variants among the proband, sister, and parents was confirmed by Sanger sequencing (Supplemental Figure 3).

Identification of compound heterozygous variants in the GINS4 gene by wholeFigure 2

Identification of compound heterozygous variants in the GINS4 gene by whole-exome sequencing. (A) Pedigree of the family denoting variants of GINS4. (B) GINS4 protein 3D structure prediction showing α-helices at N-terminus and B-strands at C-terminus (variants labeled in violet). (C) Schematic representation of GINS4 (SLD5) protein and variants mapping at the C-terminal domain. (D) Multiple protein sequence analysis using ClustalW shows evolutionary conservation of the C-terminal domain of GINS4. Conserved amino acids are highlighted in light blue. The position of both variants is indicated.

Bioinformatic analyses suggested that these variants are damaging and pathogenic. Specifically, both are in a highly conserved region (Figure 2D) and very rare; only 7 V171L and no Q191X alleles are reported in The Genome Aggregation Database (gnomAD v3.1.1). Using Combined Annotation Dependent Depletion (CADD) scores (30), the variants are predicted as damaging, with values of 26.4 (V171L) and 39 (Q191X) (for GINS4-specific mutation significance cutoff score, MSC of 5.720) (31). Moreover, a gene damage index (GDI) prediction of 1.022 suggests that GINS4 is likely to be a disease-causing gene (32). A complete list of all prioritized variants identified is provided in Supplemental Table 2. Given the genetic model of inheritance, fulfillment of bioinformatic criteria, and literature demonstrating a connection between the DNA replicative helicase and NKD, we considered GINS4 a potential candidate disease gene.

Variants in GINS4 affect GINS complex expression and assembly. GINS4 is a small protein of 26 kDa consisting of 223 amino acids. To evaluate the potentially damaging impact of the variants, we first assessed protein expression. EBV-transformed B lymphoblastoid cell lines (BLCL) derived from peripheral blood of both the proband and his sister consistently expressed 15%–20% of the GINS4 protein found in the BLCL of HDs, underscoring the combined damaging effects of the variants that lead to the destabilization of the protein (Figure 3A). Both variants map at the C-terminal domain, and the premature stop codon Q191X maps 4 nucleotides upstream of the last exon-exon junction, which, according to the 50–55 nucleotide rule, should give rise to transcripts able to escape nonsense-mediated decay (NMD) (33). The truncated allele is predicted to result in a shorter protein of 23 kDa and was detectable by Western blot of BLCL lysates at the expected molecular size (Figure 3A).

V171L and Q191X destabilize the expression of GINS4 and other GINS proteinsFigure 3

V171L and Q191X destabilize the expression of GINS4 and other GINS proteins and impair GINS complex assembly. (A) GINS4 and GINS1 protein expression in BLCL derived from siblings and parents compared with those from 2 healthy donors (HD). Relative GINS4 protein expression of family members and 2 HDs from 3 independent experiments. (B) GINS complex 3D structure showing the position of V171L and Q191X variants. (C) GINS2 and GINS3 protein expression in BLCL derived from siblings and parents compared with 2 HD. One representative experiment of 3 independent experiments is shown (D) HEK293T cells were transiently transfected with Myc-GINS4 WT, Myc-GINS4-V171L, or Myc-GINS4 Q191X plasmids. After 48 hours, whole cellular extracts were immunoprecipitated with anti-MYC, and blots were probed for GINS1, GINS2, GINS3, and MYC. Data are representative of 3 independent experiments.

Expression of Myc-GINS4 WT and Myc-GINS4 Q191X plasmids in HEK293T cells confirmed the specificity of the antibody used to detect GINS4 in BLCL and the ability to detect the shorter mutant isoform (Supplemental Figure 4A). To further evaluate the possibility that the stop codon allele can partially escape NMD, we sequenced the cDNA samples derived from the individual-derived BLCLs, which confirmed the presence of the stop codon transcript in both siblings and the mother, demonstrating at least the ongoing presence of the transcript for the truncation variant in physiologic material (Supplemental Figure 4B). In addition, the missense variant V171L appeared partially unstable at the protein level, as both siblings had consistent protein expression of the full-length variant ranging from 15% to 20%, lower than the expected 50% (Figure 3A). In line with this observation, the father carrying the single V171L heterozygous variant expressed 70%–75% of the protein (Figure 3A).

GINS4 is part of the GINS complex (Figure 3B), which is comprised of 4 subunits that are tightly associated and regulated (34). Crystal structures and electron microscopy of the GINS complex define a trapezoidal tetramer held by intersubunit hydrophobic bonds, in which GINS4 is directly bound to GINS2 through both its N-terminal and C-terminal domains and to GINS1 and GINS3 through its C-terminal domain. The N-terminal interaction between GINS2 and GINS4 is the most extensive interface in the complex (35). The crystal structure further indicates that the C-terminal residues of GINS4 are required for the assembly of the GINS core complex (36). Due to precedence for the level of GINS components impacting the expression of other GINS proteins (14, 37), we assessed the levels of GINS proteins by Western blot in BLCLs. Substantively lower levels of GI

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