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NFKB-ACTIVATING PROTEIN; NKAP

NFKB-ACTIVATING PROTEIN; NKAP

HGNC Approved Gene Symbol: NKAPCytogenetic location: Xq24 Genomic coordinates (GRCh38): X:119,920,671-119,943,750 (from NCBI)▼ DescriptionNKAP is a highly co...

HGNC Approved Gene Symbol: NKAP

Cytogenetic location: Xq24 Genomic coordinates (GRCh38): X:119,920,671-119,943,750 (from NCBI)

▼ Description
NKAP is a highly conserved protein with roles in transcriptional repression, T-cell development, maturation and acquisition of functional competency and maintenance and survival of adult hematopoietic stem cells, and RNA splicing and processing (summary by Burgute et al., 2014).

▼ Cloning and Expression
Using a yeast 2-hybrid screen of a B-cell cDNA library to identify RIP (RIPK1; 603453)-interacting proteins, followed by database and PCR analyses, Chen et al. (2003) cloned NKAP. The predicted 415-amino acid protein is highly charged and basic. It is highly conserved in evolution and shares 90% amino acid identity with mouse Nkap. Western blot analysis showed expression of a 52-kD protein in transfected human embryonic kidney 293 (HEK293) cells. Immunofluorescence microscopy demonstrated nuclear expression of endogenous and overexpressed NKAP in HEK293 cells.

Burgute et al. (2014) stated that mouse and human NKAP contains an N-terminal RS motif, followed by a highly basic domain and a C-terminal DUF926 domain. Western blot analysis showed that the mouse Nkap protein had an apparent molecular mass of 60 kD and was expressed ubiquitously, with highest expression in spleen, skin, testis, kidney, and lung. RT-PCR and immunofluorescence analyses revealed that Nkap was highly expressed in mouse brain from embryo to adult. Immunofluorescence analysis of HeLa cells showed that endogenous NKAP localized exclusively to nucleus in a punctuated pattern resembling nuclear speckles. Deletion analysis using mouse Nkap showed that the basic region was required for nuclear speckle localization.

In human cells, Fiordaliso et al. (2019) confirmed that NKAP localized to the nucleus in a nuclear speckled pattern, consistent with nucleoplasmic granules enriched in mRNA processing proteins, such as splicing factors (Burgute et al., 2014).

▼ Gene Function
Chen et al. (2003) found that although NKAP interacted with RIP in yeast 2-hybrid assays, the proteins did not interact in transfected 293 cells. Reporter gene analysis showed that NKAP activated NFKB (see 164011), but not AP1 (165160), in a dose-dependent manner. Knockdown of NKAP by antisense RNA inhibited TNF (191160)- and IL1 (see 147760)-induced NFKB activation. Chen et al. (2003) concluded that NKAP is a nuclear regulator of NFKB activation pathways.

Pajerowski et al. (2009) identified NKAP through its ability to complement a mutant human T-cell line. Transfection experiments showed that NKAP coimmunoprecipitated with CIR (605228) and with HDAC3 (605166), but not with CSL (CSHL1; 603515) or with other HDACs. The interaction with HDAC3 required the C terminus of NKAP. Luciferase and chromatin immunoprecipitation analysis revealed that NKAP functioned as a transcriptional repressor at a NOTCH (see 190198)-regulated promoter. Mutation analysis showed that neither HDAC3-binding nor CIR-binding activity alone was sufficient for NKAP to function as a repressor of NOTCH-mediated transcription. Flow cytometric analysis showed an inverse relationship between Nkap and Deltex-1 (DTX1; 602582) expression throughout T-cell development in mice. Conditional Nkap deficiency blocked development of alpha-beta T cells at the DN3 stage and led to increased expression of Notch target genes. Pajerowski et al. (2009) concluded that NKAP functions as a transcriptional repressor that acts on NOTCH target genes and is required for alpha-beta T-cell development.

Burgute et al. (2014) found that overexpression of mouse Nkap in HeLa cells dramatically altered nuclear speckle organization and increased nuclear speckle number. Pull-down experiments in transfected HeLa cells showed that mouse Nkap interacted with RNA-binding proteins, RNA helicases, and splicing factors, including FUS (137070). Interaction of mouse Nkap and Fus required the RS domain of Nkap and the RGG1 and RGG3 domains of Fus. In vitro splicing assays revealed that Nkap bound both spliced mRNA and unspliced pre-mRNA. Genomewide crosslinking and immunoprecipitation-sequencing analysis in HEK293T cells showed interaction of NKAP with diverse primary RNA transcripts, particularly with exonic regions of pre-mRNA. NKAP also bound various noncoding RNAs, including U1 (see 180680), U4, and U5 (see 180691) small nuclear RNAs. Knockdown of NKAP in HEK293T cells led to an increase in pre-mRNA percentage. The authors concluded that NKAP is a nuclear speckle protein with roles in RNA splicing and processing.

▼ Mapping
Stumpf (2020) mapped the NKAP gene to chromosome Xq24 based on an alignment of the NKAP sequence (GenBank BC015354) with the genomic sequence (GRCh38).

▼ Molecular Genetics
In a German man with Hackmann-Di Donato-type X-linked syndromic intellectual disability (MRXSHD; 301039), Hackmann et al. (2016) identified a hemizygous missense mutation in the NKAP gene (R333Q; 300766.0001). The mutation, which was found by next-generation exome sequencing of the X chromosome, was inherited from the unaffected mother. Functional studies of the variant and studies of patient cells were not performed. The patient was part of a cohort of individuals with a phenotype reminiscent of Lujan-Fryns syndrome (MRXSLF; 309520).

In 10 patients from 8 unrelated families with MRXSHD, including the patient reported by Hackmann et al. (2016), Fiordaliso et al. (2019) identified 5 different hemizygous missense variants in the NKAP gene (300766.0001-300766.0005). The R333Q mutation was identified in 6 patients. The mutations, which were found by exome sequencing, occurred de novo in 3 patients and presumed de novo in 1 patient, was maternally inherited in 3 patients from 2 families, and occurred de novo in 2 brothers, consistent with maternal germline mosaicism. All mutations occurred in exons 8 or 9 and affected the C-terminal HDAC3 (605166) transcriptional regulation/repression domain. Studies of lymphoblastoid cell lines derived from patients with 2 of the mutations (R333Q and R361Q) showed mRNA and protein levels as well as intracellular nuclear localization patterns similar to wildtype. Transcriptome analysis of these patient-derived cells showed a uniquely disrupted profile, with 455 upregulated and 721 downregulated genes compared to controls. Differentially expressed genes (DEGs) included HES1 (139605) and JAG1 (601920), which play a role in NOTCH signaling pathways, as well as dysregulation of genes involved in the extracellular matrix. Additional findings included lack of a change in major splicing patterns and downregulation of long genes with a higher number of exons. Fiordaliso et al. (2019) concluded that NKAP has a critical role in transcriptional regulation during development, particularly for neurons and connective tissue.

▼ Animal Model
Hsu et al. (2011) generated conditional Nkap-knockout mice, which exhibited normal thymic cellularity but developed a severe defect in peripheral T-cell numbers. The conditional Nkap-knockout mice had defective expression of Qa2, a mouse marker of T-cell maturation and functional competence. Peripheral naive Cd4 cells from conditional Nkap-knockout mice were functionally immature recent emigrants from thymus. Expression of a Bcl2 (151430) transgene failed to rescue the T-cell maturation deficiency, indicating that the maturation defect was not due to a failure of cell survival. Hsu et al. (2011) concluded that NKAP is required for T-cell maturation and the acquisition of functional competency.

Fiordaliso et al. (2019) found that heterozygous knockdown of nkap in zebrafish did not result in significant abnormalities compared to wildtype. However, homozygous mutant animals showed striking defects in early development, including edema in the eye, intestinal tracts, and heart, curvature of the notochord, and central nervous system necrosis. All homozygous mutant animals lacked a heartbeat at 4 days postfertilization (dpf) and began to decompose. The generated mutation produced a C-terminally truncated protein, indicating that this region is essential for proper development.

▼ ALLELIC VARIANTS ( 5 Selected Examples):

.0001 INTELLECTUAL DEVELOPMENTAL DISORDER, X-LINKED, SYNDROMIC, HACKMANN-DI DONATO TYPE
NKAP, ARG333GLN
In a German man with Hackmann-Di Donato-type X-linked syndromic intellectual disability (MRXSHD; 301039), Hackmann et al. (2016) identified a hemizygous c.998G-A transition in the NKAP gene, resulting in an arg333-to-gln (R333Q) substitution. The mutation, which was found by next generation exome sequencing of the X-chromosome, was inherited from the unaffected mother. functional studies of the variant and studies of patient cells were not performed. The patient was part of a cohort of individuals with a phenotype reminiscent of Lujan-Fryns syndrome (MRXSLF; 309520).

In 5 additional males from 3 unrelated families with MRXSHD, Fiordaliso et al. (2019) identified a hemizygous R333Q mutation in exon 8 of the NKAP gene. The substitution occurred at a highly conserved residue in the C-terminal HDAC3 (605166) transcriptional regulation/repression domain. The mutation, which was found by exome sequencing, was not present in the gnomAD database. In 1 family, the mutation occurred de novo, likely from maternal gonadal mosaicism, whereas it was maternally inherited in another family. The families were of various ethnic origins, including Japanese and Slovak. Studies of lymphoblastoid cell lines derived from patients showed mRNA and protein levels as well as intracellular nuclear localization patterns similar to wildtype.

.0002 INTELLECTUAL DEVELOPMENTAL DISORDER, X-LINKED, SYNDROMIC, HACKMANN-DI DONATO TYPE
NKAP, ARG330CYS
In a 10-year-old Japanese boy (patient 1) with Hackmann-Di Donato-type X-linked syndromic intellectual developmental disorder (MRXSHD; 301039), Fiordaliso et al. (2019) identified a de novo hemizygous c.988C-T transition in exon 8 of the NKAP gene, resulting in an arg330-to-cys (R330C) substitution at a highly conserved residue in the C-terminal HDAC3 (605166) transcriptional regulation/repression domain. The mutation, which was found by exome sequencing, was not present in the gnomAD database. Functional studies of the variant and studies of patient cells were not performed.

.0003 INTELLECTUAL DEVELOPMENTAL DISORDER, X-LINKED, SYNDROMIC, HACKMANN-DI DONATO TYPE
NKAP, ARG330HIS
In a 4-year-old Korean boy (patient 2) with Hackmann-Di Donato-type X-linked syndromic intellectual developmental disorder (MRXSHD; 301039), Fiordaliso et al. (2019) identified a de novo hemizygous c.989G-A transition in exon 8 of the NKAP gene, resulting in an arg330-to-his (R330H) substitution at a highly conserved residue in the C-terminal HDAC3 (605166) transcriptional regulation/repression domain. The mutation, which was found by exome sequencing, was not present in the gnomAD database. Functional studies of the variant and studies of patient cells were not performed.

.0004 INTELLECTUAL DEVELOPMENTAL DISORDER, X-LINKED, SYNDROMIC, HACKMANN-DI DONATO TYPE
NKAP, ILE337THR
In a 19-year-old man (patient 9) from the UK with Hackmann-Di Donato-type X-linked syndromic intellectual developmental disorder (MRXSHD; 301039), Fiordaliso et al. (2019) identified a de novo hemizygous c.1010T-C transition in exon 8 of the NKAP gene, resulting in an ile337-to-thr (I337T) substitution at a highly conserved residue in the C-terminal HDAC3 (605166) transcriptional regulation/repression domain. The mutation, which was found by exome sequencing, was not present in the gnomAD database. Functional studies of the variant and studies of patient cells were not performed.

.0005 INTELLECTUAL DEVELOPMENTAL DISORDER, X-LINKED, SYNDROMIC, HACKMANN-DI DONATO TYPE
NKAP, ARG361GLN
In a 16-year-old boy (patient 10) from the US with Hackmann-Di Donato-type X-linked syndromic intellectual developmental disorder (MRXSHD; 301039), Fiordaliso et al. (2019) identified a presumably de novo hemizygous c.1082G-A transition in exon 9 of the NKAP gene, resulting in an arg361-to-gln (R361Q) substitution at a highly conserved residue in the C-terminal HDAC3 (605166) transcriptional regulation/repression domain. The mutation, which was found by exome sequencing, was not present in the gnomAD database. The mutation was not present in the mother; the father was not tested. Studies of lymphoblastoid cell lines derived from the patient showed mRNA and protein levels as well as intracellular nuclear localization patterns similar to wildtype.

Tags: Xq24