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

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.

Homozygous DBF4 mutation as a cause of severe congenital neutropenia

“…We report a homozygous mutation in DBF4 associated with mild facial dysmorphism, growth retardation, mild intellectual disability and severe congenital neutropenia. This phenotype shows both significant overlap with previously reported DNA replication-associated syndromes, especially GINS1 and GINS4 deficiency 3,10,20,21 , and also distinct immunological features. For example, in functional DBF4, GINS1 and GINS4 deficiency T and B cells seem to be relatively spared, while deficiencies in subunits of the replicative DNA polymerases ε and δ are associated with a combined immunodeficiency without neutropenia 7,8,[10][11][12]14,20,21 .…”

“…A potential explanation for the qualitative model of cellular manifestations of DNA replicationassociated syndromes is the biochemical impact of deficiency. In contrast to GINS1 deficiency, both functional DBF4-and GINS4-deficient cells did not show evidence of increased DNA damage, arguing that this is not a prerequisite for the neutropenia phenotype 10,20 . Our experiments indicate that the DBF4 K209N variant has lower CDC7 binding capacity than the J o u r n a l P r e -p r o o f 19 WT protein, but we found evidence of lower MCM2 DDK-specific phosphorylation only at S139, and not at S40/41, in unchallenged fibroblasts.…”

“…The granulocyte differentiation arrest in GINS1 deficiency is characterized by the accumulation of both promyelocytes and, unlike our patient, myelocytes in the bone marrow, and was associated with only few infections and intact emergency granulopoiesis 10 . Similarly, the GINS4-deficient patients only required intermittent G-CSF treatment 20 , arguing for a more severe congenital neutropenia phenotype in the DBF4 patient. Other DNA replicationassociated syndromes do not present with neutropenia, suggesting this is a gene-specific phenotype 3,21 .…”

“…Mutations in more than 20 DNA replication-associated genes cause monogenic disease which often present with developmental defects and perturbed growth, but also differentially impact specific tissues and cell types 3 . Mutations in thirteen DNA replication factors (REQCL4, MCM4, POLE1, POLE2, POLA1, GINS1, POLD1, POLD2, MCM10, TOP2B, PRIM1, RPA1, GINS4) present with a convergent phenotype of perturbed growth, facial anomalies and variable immune cell defects [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21] . While immune defects are shared among multiple DNA replication-associated syndromes, only GINS1 and GINS4 deficiency are associated with congenital neutropenia, demonstrating a gene-specific as well as cell typespecific basis for sensitivity 3,10,21 .…”

iPSC-based modeling of helicase deficiency reveals impaired cell proliferation and increased apoptosis after NK cell lineage commitment

“…Previous studies have shown that CD34 + cells with helicase variants have impaired NK cell maturation in in vitro differentiation assays, which mirrors patient phenotypes in vivo (10, 12). Other studies have relied upon primary cell lines and PBMCs for studying functional defects in NK cells, thus limiting understanding of the full spectrum of NKD pathogenesis (9–12).…”