Alternative titles; symbols
HGNC Approved Gene Symbol: PNKP
SNOMEDCT: 719981005;
Cytogenetic location: 19q13.33 Genomic coordinates (GRCh38) : 19:49,861,204-49,867,576 (from NCBI)
| Location | Phenotype |
Phenotype MIM number |
Inheritance |
Phenotype mapping key |
|---|---|---|---|---|
| 19q13.33 | ?Charcot-Marie-Tooth disease, type 2B2 | 605589 | Autosomal recessive | 3 |
| Ataxia-oculomotor apraxia 4 | 616267 | Autosomal recessive | 3 | |
| Microcephaly, seizures, and developmental delay | 613402 | Autosomal recessive | 3 |
The PNKP gene encodes a polynucleotide kinase 3-prime phosphatase, which catalyzes the 5-prime phosphorylation of nucleic acids and also has an associated 3-prime phosphatase activity, predictive of an important function in DNA repair following ionizing radiation or oxidative damage (Jilani et al., 1999).
By searching sequence databases using 3 tryptic peptides from a bovine 60-kD polypeptide that correlated with 5-prime DNA kinase and 3-prime phosphatase activities, Jilani et al. (1999) identified human and murine EST clones encoding PNKP. The predicted 57.1-kD, 521-amino acid PNKP protein contains a putative ATP-binding site and a potential 3-prime phosphatase domain with similarity to L-2 haloacid dehalogenases. Database searches identified possible homologs in C. elegans, S. pombe, and Drosophila. Northern blot analysis detected a 2-kb transcript in 8 human tissues, with highest expression in spleen and testis, and lowest expression in small intestine. A second signal of about 7.5 kb was also observed, with highest intensity in spleen. A glutathione S-transferase-PNKP fusion protein displayed 5-prime DNA kinase and 3-prime phosphatase activities. The authors stated that PNKP was the first gene identified for a DNA-specific kinase from any organism. PNKP expression partially rescued the sensitivity to oxidative damaging agents of the E. coli DNA repair-deficient 'xth nfo' double mutant. Furthermore, PNKP gene function restored termini suitable for DNA polymerase, consistent with in vivo removal of 3-prime phosphate groups, facilitating DNA repair.
Karimi-Busheri et al. (1999) purified the PNKP enzyme, which they called PNK, from HeLa cells and obtained the amino acid sequences for several tryptic fragments by mass spectrometry. By searching an EST database, the sequences were matched with an incomplete human cDNA clone, which was used as a probe to retrieve the 5-prime end of the cDNA sequence from a HeLa cell cDNA library. The complete cDNA was expressed in E. coli, and the recombinant protein was shown to possess the kinase and phosphatase activities. Comparison of PNKP with other sequenced proteins identified a P-loop motif, indicative of an ATP-binding domain, and a second motif associated with several different phosphatases. The authors found reasonable sequence similarity to putative open reading frames in the genomes of C. elegans and S. pombe, but similarity to bacteriophage T4 polynucleotide kinase is limited to the kinase and phosphatase domains. Northern blot analysis revealed a major transcript of approximately 2.3 kb and a minor transcript of approximately 7 kb, with expression levels higher in pancreas, heart, and kidney than in brain, lung, and liver. Confocal microscopy of human A549 cells indicated that PNKP resides predominantly in the nucleus.
By in situ hybridization, Shen et al. (2010) found that human and mouse PNKP mRNA were expressed in both dividing neuronal precursors in the cerebral cortical ventricular zone, and in postmitotic neurons of the cortical plate.
The PNKP gene contains 17 exons (Shen et al., 2010).
By FISH, Jilani et al. (1999) mapped the PNKP gene to chromosome 19q13.3-q13.4. Karimi-Busheri et al. (1999) mapped the PNKP gene to 19q13.4 using FISH.
Microcephaly, Seizures, and Developmental Delay
By genomewide linkage analysis followed by candidate gene sequencing of a region on chromosome 19q13 in families with microcephaly, seizures, and developmental delay (MCSZ; 613402), Shen et al. (2010) identified homozygous or compound heterozygous mutations in the PNKP gene (605610.0001-605610.0004), resulting in loss of protein function. In most patients, the phenotype was characterized by onset of seizures in infancy, consistent with a developmental and epileptic encephalopathy (DEE10). Cells from 1 affected individual showed sensitivity to irradiation in culture, reflecting a deficiency in nonhomologous end-joining. In addition, patients' cells were significantly impaired in their ability to repair hydrogen-peroxide induced free radical DNA damage, as well as showing delayed ability to repair camptothecin-induced damage compared to controls. RNA interference of Pnkp in dissociated mouse neurons in culture resulted in increased apoptosis of both precursor and differentiated neurons. Shen et al. (2010) suggested a role for PNKP in several DNA repair pathways.
In 2 Dutch brothers, born of consanguineous parents, with a somewhat protracted course of MCSZ, Poulton et al. (2013) identified a homozygous truncating mutation in the PNKP gene (605610.0002). Patient fibroblasts showed increased susceptibility under stress conditions compared to controls. The same mutation had been found by Shen et al. (2010) in patients with a more severe epilepsy phenotype.
Ataxia-Oculomotor Apraxia 4
In 11 patients from 8 unrelated Portuguese families with autosomal recessive ataxia-oculomotor apraxia-4 (AOA4; 616267), Bras et al. (2015) identified homozygous or compound heterozygous mutations in the PNKP gene (see, e.g., 605610.0002 and 605610.0005-605610.0008). The mutations, which were found by a combination of homozygosity mapping and exome sequencing, segregated with the disorder in all families with available samples. Functional studies of the variants were not performed. The disorder was characterized by onset of symptoms in the first decade and rapid progression; most patients became wheelchair-bound in the second or third decade.
Charcot-Marie-Tooth Disease Type 2B2
In a 17-year-old Brazilian boy with Charcot-Marie-Tooth disease type 2B2 (CMT2B2; 605589), Pedroso et al. (2015) identified a homozygous in-frame deletion in the PNKP gene (Thr408del; 605610.0007). The mutation, which was found by whole-exome sequencing, was heterozygous in the unaffected parents. In vitro functional expression studies showed that patient fibroblasts were more sensitive to genotoxic chemical injury compared to control fibroblasts. Patient cells showed increased apoptosis, increased caspase-3 (CASP3; 600636) activity, a disruption of cell cycle dynamics, and evidence of DNA damage. The findings indicated that the mutation rendered the cells unable to efficiently repair DNA for both base-excision repair (BER) and nonhomologous end-joining (NHE) pathways.
In affected members of a large multigenerational consanguineous Costa Rican family with CMT2B2, originally reported by Leal et al. (2001), Leal et al. (2018) identified a homozygous nonsense mutation in the PNKP gene (Q517X; 605610.0009). The mutation, which was found by exome sequencing and confirmed by Sanger sequencing, segregated with the disorder in the family. Analysis of the PNKP coding region in 5 additional Costa Rican probands with a similar phenotype showed that all were compound heterozygous for the Q517X mutation and a Thr408del (605610.0007) mutation. Molecular modeling predicted a damaging effect for the Q517X mutation, although functional studies and studies of patient cells were not performed. Leal et al. (2018) concluded that the mutant protein would lack an important functional C-terminal domain, causing increased DNA damage with subsequent cell death.
In 7 affected individuals from 3 consanguineous Arab Palestinian families with microcephaly, seizures, and developmental delay (MCSZ; 613402), Shen et al. (2010) identified a homozygous c.975G-A transition in exon 11 of the PNKP gene, resulting in a glu326-to-lys (E326K) substitution in a highly conserved residue that is part of the seventh alpha-helix near the phosphatase domain. Lymphocytes derived from affected individuals showed decreased PNKP expression. The mutation was not found in 280 control chromosomes. The phenotype was characterized by onset of refractory seizures in infancy, consistent with a developmental and epileptic encephalopathy (DEE).
In affected members of 2 unrelated families with microcephaly, seizures, and developmental delay (MCSZ; 613402), Shen et al. (2010) identified a homozygous 17-bp duplication (c.1250_1266dup) in exon 14 of the PNKP gene, resulting in a frameshift and premature termination. The families were of Saudi Arabian and Turkish descent, respectively. Lymphocytes derived from affected individuals showed decreased PNKP expression. Two additional unrelated European American families with the disorder were found to be compound heterozygous for the 17-bp duplication and another pathogenic mutations in the PNKP gene: L176F (605610.0003) and a 17-bp deletion (605610.0004). Despite their diverse ancestries, haplotype analysis suggested a common founder for the 17-bp duplication, which was not found in 1,080 Middle Eastern or European control chromosomes. The phenotype was characterized by onset of refractory seizures in infancy, consistent with a developmental and epileptic encephalopathy (DEE).
Poulton et al. (2013) identified the same homozygous 17-bp duplication in the PNKP gene in 2 Dutch brothers, born of consanguineous parents, with early childhood onset of a neurodegenerative disorder. The patients had global developmental delay from infancy as well as progressive and severe microcephaly, but seizures were not as severe as those reported by Shen et al. (2010). These patients developed seizures at 12 months and 2.5 years of age. Both Dutch boys also developed progressive cerebellar atrophy and a sensorimotor peripheral neuropathy, resulting in loss of independent ambulation in childhood or adolescence. Poulton et al. (2013) emphasized the neurodegenerative phenotype of these patients and suggested that it was distinct from that reported by Shen et al. (2010). Patient fibroblasts showed increased susceptibility under stress conditions compared to controls.
In 2 Portuguese sibs with ataxia-oculomotor apraxia-4 (AOA4; 616267), Bras et al. (2015) identified compound heterozygous mutations in the PNKP gene: the same 17-bp duplication in the PNKP gene identified by Shen et al. (2010) and Poulton et al. (2013), and G375W (605610.0005). Bras et al. (2015) referred to the 17-bp duplication as c.1253_1269dup, resulting in a frameshift and premature termination (Thr424GlyfsTer49) in the kinase domain. Functional studies of the variants were not performed.
In affected members of a European American family with microcephaly, seizures, and developmental delay (MCSZ; 613402), Shen et al. (2010) identified compound heterozygosity for 2 mutations in the PNKP gene: a 526C-T transition in exon 5, resulting in a leu176-to-phe (L176F) substitution in the phosphatase domain, and a 17-bp duplication (605610.0002). The L176F mutation was not found in 280 control chromosomes. The phenotype was characterized by onset of refractory seizures in infancy, consistent with a developmental and epileptic encephalopathy (DEE).
In affected members of a European American family with microcephaly, seizures, and developmental delay (MCSZ; 613402), Shen et al. (2010) identified compound heterozygosity for 2 mutations in the PNKP gene: a 17-bp deletion in intron 15, resulting in disruption of proper mRNA splicing, and a 17-bp duplication (605610.0002). RT-PCR analysis showed that the deletion resulted in the skipping of exon 15 and low concentrations of normal protein. However, the phenotype in the 2 affected individuals was slightly less severe than observed in patients with other PNKP mutations, suggesting that there may be residual protein activity. The 17-bp deletion was not found in 280 control chromosomes.
In 6 patients from 4 Portuguese families with ataxia-oculomotor apraxia-4 (AOA4; 616267), Bras et al. (2015) identified a homozygous c.1123G-T transversion in the PNKP gene, resulting in a gly375-to-trp (G375W) substitution at a highly conserved residue in a potential ATP nucleotide-binding domain in the kinase region. The mutation, which was found by a combination of homozygosity mapping and exome sequencing, was confirmed by Sanger sequencing and filtered against the dbSNP (build 137) database and an in-house database of over 2,000 samples. Affected individuals from 2 additional families were compound heterozygous for G375W and a frameshift mutation (605610.0002 and 605610.0006, respectively). All mutations segregated with the disorder in the families. Functional studies of the variants were not performed.
In a Portuguese girl, born of consanguineous parents, with ataxia-oculomotor apraxia-4 (AOA4; 616267), Bras et al. (2015) identified compound heterozygous mutations in the PNKP gene: a 5-bp insertion (c.1322_1323insAGCCG), resulting in a frameshift and premature termination (Gly442AlafsTer27), and G375W (605610.0005). The mutations were found by Sanger sequencing; family segregation studies and functional studies were not performed.
Ataxia-Oculomotor Apraxia 4
In 2 unrelated Portuguese patients with ataxia-oculomotor apraxia-4 (AOA4; 616267), Bras et al. (2015) identified a heterozygous 3-bp deletion (c.1221_1223del) in the PNKP gene, resulting in the deletion of residue thr408 (Thr408del). Each patient had a truncating mutation in the PNKP gene on the other allele (see, e.g., 605610.0008). The mutations segregated with the disorder in the families; functional studies of the variants were not performed.
Charcot-Marie-Tooth Disease, Axonal, Type 2B2
In a 17-year-old Brazilian boy with Charcot-Marie-Tooth disease type 2B2 (CMT2B2; 605589), Pedroso et al. (2015) identified a homozygous Thr408del mutation in the PNKP gene. The mutation, which was found by whole-exome sequencing, was heterozygous in the unaffected parents. In vitro functional expression studies showed that patient fibroblasts were more sensitive to genotoxic chemical injury compared to control fibroblasts. Patient cells showed increased apoptosis, increased caspase-3 (CASP3; 600636) activity, a disruption of cell cycle dynamics, and evidence of DNA damage. The findings indicated that the mutation rendered the cells unable to efficiently repair DNA for both base-excision repair (BER) and nonhomologous end-joining (NHE) pathways.
For discussion of the Thr408del mutation in the PNKP gene that was found in compound heterozygous state in 5 unrelated Costa Rican probands with CMT2B2 by Leal et al. (2018), see 605610.0009. Leal et al. (2018) stated that the Thr408del variant, which occurs in exon 14, was present in the gnomAD database (rs770849181) at a low frequency (15 of 214,338 alleles).
In a Portuguese girl with ataxia-oculomotor apraxia-4 (AOA4; 616267), Bras et al. (2015) identified compound heterozygous mutations in the PNKP gene: an 8-bp insertion (c.1549_1550ins), resulting in a frameshift and premature termination (Gln517LeufsTer24), and Thr408del (605610.0007). The mutations segregated with the disorder in the family; functional studies of the variants were not performed.
In affected members of a large multigenerational consanguineous Costa Rican family with Charcot-Marie-Tooth disease type 2B2 (CMT2B2; 605589), originally reported by Leal et al. (2001), Leal et al. (2018) identified a homozygous c.1549C-T transition in the last exon (exon 17) of the PNKP gene, resulting in a gln517-to-ter (Q517X) substitution. The mutation, which was found by exome sequencing and confirmed by Sanger sequencing, segregated with the disorder in the family. The variant has a low frequency in the gnomAD database (18 of 245,644 alleles). Previously, the disorder in this family was erroneously attributed to a homozygous variant in the MED25 gene (A335V; 610197.0001), which maps to the same region as PNKP. The MED25 and PNKP variants both fully segregated with the disorder in the family and were shown to be present on the same haplotype, suggesting an ancestral founder haplotype. Analysis of the PNKP coding sequencing in 5 additional Costa Rican probands with a similar phenotype showed that all were compound heterozygous for the Q517X mutation and a Thr408del (605610.0007) mutation. These patients also carried the ancestral haplotype with the MED25 variant. Molecular modeling predicted a damaging effect for the Q517X mutation, although functional studies and studies of patient cells were not performed. Leal et al. (2018) concluded that the mutant protein would lack an important functional C-terminal domain, causing increased DNA damage with subsequent cell death.
Bras, J., Alonso, I., Barbot, C., Costa, M. M., Darwent, L., Orme, T., Sequeiros, J., Hardy, J., Coutinho, P., Guerreiro, R. Mutations in PNKP cause recessive ataxia with oculomotor apraxia type 4. Am. J. Hum. Genet. 96: 474-479, 2015. [PubMed: 25728773] [Full Text: https://doi.org/10.1016/j.ajhg.2015.01.005]
Jilani, A., Ramotar, D., Slack, C., Ong, C., Yang, X. M., Scherer, S. W., Lasko, D. D. Molecular cloning of the human gene, PNKP, encoding a polynucleotide kinase 3-prime-phosphatase and evidence for its role in repair of DNA strand breaks caused by oxidative damage. J. Biol. Chem. 274: 24176-24186, 1999. [PubMed: 10446192] [Full Text: https://doi.org/10.1074/jbc.274.34.24176]
Karimi-Busheri, F., Daly, G., Robins, P., Canas, B., Pappin, D. J. C., Sgouros, J., Miller, G. G., Fakhrai, H., Davis, E. M., Le Beau, M. M., Weinfeld, M. Molecular characterization of a human DNA kinase. J. Biol. Chem. 274: 24187-24194, 1999. [PubMed: 10446193] [Full Text: https://doi.org/10.1074/jbc.274.34.24187]
Leal, A., Bogantes-Ledezma, S., Ekici, A. B., Uebe, S., Thiel, C. T., Sticht, H., Berghoff, M., Berghoff, C., Morera, B., Meisterernst, M., Reis, A. The polynucleotide kinase 3-prime-phosphatase (PNKP) is involved in Charcot-Marie-Tooth disease (CMT2B2) previously related to MED25. Neurogenetics 19: 215-225, 2018. [PubMed: 30039206] [Full Text: https://doi.org/10.1007/s10048-018-0555-7]
Leal, A., Morera, B., Del Valle, G., Heuss, D., Kayser, C., Berghoff, M., Villegas, R., Hernandez, E., Mendez, M., Hennies, H. C., Neundorfer, B., Barrantes, R., Reis, A., Rautenstrauss, B. A second locus for an axonal form of autosomal recessive Charcot-Marie-Tooth disease maps to chromosome 19q13.3. Am. J. Hum. Genet. 68: 269-274, 2001. [PubMed: 11112660] [Full Text: https://doi.org/10.1086/316934]
Pedroso, J. L., Rocha, C. R. R., Macedo-Souza, L. I., De Mario, V., Marques, W., Jr., Barsottini, O. G. P., Oliveira, A. S. B., Menck, C. F. M., Kok, F. Mutation in PNKP presenting initially as axonal Charcot-Marie-Tooth disease. Neurol. Genet. 1: e30, 2015. Note: Electronic Article. [PubMed: 27066567] [Full Text: https://doi.org/10.1212/NXG.0000000000000030]
Poulton, C., Oegema, R., Heijsman, D., Hoogeboom, J., Schot, R., Stroink, H., Willemsen, M. A., Verheijen, F. W., van de Spek, P., Kremer, A., Mancini, G. M. S. Progressive cerebellar atrophy and polyneuropathy: expanding the spectrum of PNKP mutations. Neurogenetics 14: 43-51, 2013. [PubMed: 23224214] [Full Text: https://doi.org/10.1007/s10048-012-0351-8]
Shen, J., Gilmore, E. C., Marshall, C. A., Haddadin, M., Reynolds, J. J., Eyaid, W., Bodell, A., Barry, B., Gleason, D., Allen, K., Ganesh, V. S., Chang, B. S., Grix, A., Hill, R. S., Topcu, M., Caldecott, K. W., Barkovich, A. J., Walsh, C. A. Mutations in PNKP cause microcephaly, seizures and defects in DNA repair. (Letter) Nature Genet. 42: 245-249, 2010. [PubMed: 20118933] [Full Text: https://doi.org/10.1038/ng.526]