Mutations in PNKP cause microcephaly, seizures and defects in DNA repair

Abstract

Maintenance of DNA integrity is crucial for all cell types, but neurons are particularly sensitive to mutations in DNA repair genes, which lead to both abnormal development and neurodegeneration. We describe a previously unknown autosomal recessive disease characterized by microcephaly, early-onset, intractable seizures and developmental delay (denoted MCSZ). Using genome-wide linkage analysis in consanguineous families, we mapped the disease locus to chromosome 19q13.33 and identified multiple mutations in PNKP (polynucleotide kinase 3'-phosphatase) that result in severe neurological disease; in contrast, a splicing mutation is associated with more moderate symptoms. Unexpectedly, although the cells of individuals carrying this mutation are sensitive to radiation and other DNA-damaging agents, no such individual has yet developed cancer or immunodeficiency. Unlike other DNA repair defects that affect humans, PNKP mutations universally cause severe seizures. The neurological abnormalities in individuals with MCSZ may reflect a role for PNKP in several DNA repair pathways.

Figure 1. Pedigrees of MCSZ families

Family 1 represents a consanguineous Palestinian pedigree in Jordan. Family 2 shows another consanguineous Palestinian pedigree reportedly unrelated to Family 1 also in Jordan. Family 3 is also consanguineous and Palestinian but now in the USA. Family 4 is from the Kingdom of Saudi Arabia and the parents were not known to be consanguineous. Family 5 is from Turkey and the parents were not known to be related. Family 6 is of mixed European descent from the USA (German-Irish). Family 7 is also of mixed European (Swedish, Italian, Irish and English) heritage from the USA. The individuals from whom samples were obtained are labeled “DNA”. The individuals from whom we established lymphoid cell lines are labeled “Cells”. Cells and DNA were available for all Family 7 members.

Figure 2. Brain MRIs of MCSZ patients

Representative MRI images are shown from Families 4 (a, severely affected) and 7 (b, moderately affected) with aged matched controls. MRIs of severely affected patients from other families were similar to the representative images in a. Sagittal images are shown on the left (T1), axial images in the middle (T2) and coronal images on the right (T2 (a) and FLAIR (b)) with the MRI sequence noted above the image. The MRIs illustrate that despite the microencephaly (small brain), the gyral pattern is not clearly abnormal indicating absence of visible neuronal migration abnormality. The cerebellum is proportionately small compared to the cerebrum and the subpallium (basal ganglia or ventral cerebrum) is proportionately with the pallium (dorsal cerebrum). There is no evidence of atrophy or glial scarring. Bar=5 cm for both unaffected and MCSZ images.

Figure 3. PNKP mutations in MCSZ patients

(a) Four different mutations diagrammed in human PNKP genomic DNA, mRNA, and protein including domains (forkhead is FHA). The human PNKP gene consists of 17 exons shown as boxes and encodes a peptide of 521 amino acids. Filled boxes represent untranslated regions and open boxes represent coding regions. Lines connecting the exons represent introns. (b) Western blot for PNKP, the first and second lanes show a MCSZ patient (VI:3 with E326K mutation) and unaffected brother (VI:1), respectively, from Family 3. The third and fourth lanes show MCSZ patients (II:1 and 4, both with 17 bp dup and 17 bp intron 15 del) while the fifth and sixth lanes the father (I:1) and brother (II:3) both heterozygous for 17 bp intron 15 del. The band is ~60 kD (57 kD predicted size). Anti-β-actin is a loading control. (c) RT-PCR products of mRNA from members of Family 7 show the expected size from the normal copy of PNKP cDNA, 636 bp, seen in lanes 3 and 4 from non-affected carriers. The 17 bp dup results in a 653 bp long fragment seen in lanes 1, 2 and 3. The 548 bp band in lanes 1, 2, and 4 show samples with the intron 15 deletion lacking exon 15 (determined from sequencing, data not shown). A small amount of normal sized transcript is seen in lanes 1 and 2 with higher exposure (data not shown), indicating that a small amount of normal PNKP mRNA by be produced.

Figure 4. Patient derived lymphocytes show abnormal DNA repair

(a) Examples of comet assay results are shown. The intensity of the fluorescence is represented in pseudocolor while the electrical field drives damaged, loose DNA from left to right. (I) Shows a cell with 50% Tail DNA with the body of the nucleus (green) on the left and the tail of the comet derived from the damaged DNA extending to the right. (II and III) show progressively less damage, 29% and 11% Tail DNA respectively. (b) After hydrogen peroxide (H₂O₂) treatment with 0 minutes for recovery, cells show their maximum damage. Cells derived from MCSZ patients (blue, red), show significant impairment in their ability to repair DNA after hydrogen peroxide was removed, while cells derived from unaffected family members were able to repair DNA much more efficiently. (c) After camptothecin (CPT) treatment, there was also statistically significantly slower repair in cells derived from MCSZ patients compared to unaffected family members (green, purple) as well. However, after 45 minutes, the MCSZ derived cells were able to repair all CPT damage, in contrast to H₂O₂-treated cells. All cells derived from Family 7. Blue diamond and red square were from MCSZ patients with exon 14 17 bp duplication and intron 15 17 bp deletion (II:1 and II:4 respectively), green triangle were from unaffected parent heterozygous for the intron 15 17 bp deletion (I:1) and purple X from unaffected sibling with no mutation (II:2). *(P=0.05), **(P<0.005) and ***(P<0.0005). Scale bar shows 50 μm.

Figure 5. PNKP in situ hybridization

In situ hybridization of Carnegie Stage 22 human embryos (~54 postovulatory days) with anti-sense probe to human PNKP (a). Sense strand (not shown) showed no specific hybridization. Higher magnification image of developing cerebral cortex boxed area in (a) is shown in (b). Ventricular zone (VZ), containing proliferating cells, shows PNKP mRNA expression while the cell-sparse marginal zone (MZ) has no staining. Mouse E14 cerebral cortex (c) with high magnification of boxed region shown in (d) shows a similar staining pattern with high expression within the proliferating VZ and lower but maintained expression within differentiated neurons of the cortical plate (CP). (a) and (b) are in the transverse plane and (c) and (d) are coronal. Scale bars, (a) 1 mm, (b) 100 μm, (c) 150 μm, (d) 75 μm.