The polynucleotide kinase 3′-phosphatase gene (PNKP) is involved in Charcot-Marie-Tooth disease (CMT2B2) previously related to MED25

In 2001, we reported mapping of a locus for an axonal Charcot-Marie-Tooth disease with autosomal recessive pattern of inheritance to chromosome 19q13.3 (CMT2B2, MIM 605589). Affected members of a Costa Rican family (CR-P) presented with a symmetric motor and sensory neuropathy, distal muscle wasting, impaired deep tendon reflexes, and age at onset between 28 and 42 years. Electrophysiological studies revealed an axonal degenerative process (CMT2) [1]. Fine-mapping allowed refining of the critical interval to 1 Mb encompassing a total of 53 established or putative genes. An exhaustive search for the causative variant with Sanger sequencing of all exons identified a single missense variant (c.1004C>T) p.Ala335Val in the Mediator complex 25 (MED25) gene, encoding a subunit of an RNA polymerase II transcriptional regulator complex. The variant is located in a proline-rich region with high affinity for SH3 domains of the Abelson type, and we could demonstrate that it leads to a decreased binding specificity. Also, we showed that in mice and rats Med25 is coordinately expressed with Pmp22 gene, a gene involved in the Charcot-Marie-Tooth disease [2]. More recently, an individual was identified with an axonal form of CMT carrying compound heterozygous variants (p.Ala335Val and p.Pro656Thr) in MED25, although this individual also presented with additional variants in other CMT-related genes [3]. Finally, suppression of med25 in Zebrafish caused damage in the axon of peripheral neurons [3]. All these findings suggested a causative role for MED25 in CMT2.

Despite this, it was remarkable that until now no additional CMT families with variants in MED25 were identified. To exclude that other variants were missed in the initial Sanger-based analysis and given that meanwhile, high-throughput sequencing became available, we decided to perform exome sequencing in several affected members of the CR-P family. This led now to the identification of homozygous nonsense variant in the last exon of an adjacent gene encoding the polynucleotide kinase 3′-phosphatase (PNKP). Although this variant was already identified in our original screen, it was nevertheless discarded as the PNKP gene annotation used at the time included an upstream stop codon causing the variant to be mapped outside the coding sequence.

PKNP catalyzes the 5-prime phosphorylation of nucleic acids and also has an associated 3-prime phosphatase activity, an important function in DNA repair following ionizing radiation or oxidative damage [4]. It acts as a 5′-kinase/3′-phosphatase to create 5′-phosphate/3′-hydroxyl termini, which are a necessary prerequisite for ligation during repair [5]. Homozygous variants in this gene were previously linked to progressive cerebellar atrophy and polyneuropathy [6] and recently, to both severe ataxia with oculomotor apraxia 4 (AOA4, MIM 616267) or microcephaly with seizures and developmental delay (MCSZ, MIM 613402) [7]. Our findings imply that this DNA repair enzyme is involved in a broader phenotype, including a mild axonal peripheral polyneuropathy.

We provide evidence that a variant in the polynucleotide kinase 3′-phosphatase (PNKP) gene is responsible for the CMT2B2 in a large Costa Rican family (CR-P), previously associated with a variant in the subunit 25 of the Mediator complex (MED25) gene. Although all affected members of this family are homozygous both for the p.Ala335Val variant in MED25 and p.Gln517del in PNKP, we have identified five additional individuals from other Costa Rican CMT2 families, who are heterozygous for the MED25 variant (without additional MED25 variants) but compound heterozygous for two PNKP variants (p.Gln517ter/p.Thr408del). This indicates that variants on PNKP are the principal cause of the disease. PNKP is located in the original linkage interval and it was tested in our patients. Nevertheless, the c. C1549T variant was initially not considered, as the annotation of that gene was incomplete when we performed the first Sanger sequencing analysis in 2001. The variant is located in what is now recognized at PNKP’s last exon.

The PNKP variant p.Gln517ter is a rare variant with a frequency of 18/245,644 (gnomAD All), in agreement with what is expected for a deleterious mutation. Aminoacids at positions 517–521 of PNKP are very well evolutionary conserved in diverse species (Fig. 7) suggesting that C-terminal tail is critical for its function. The Gln517ter variant, homozygous for the affected individuals of the large family, causes the loss of those amino acids of the enzyme, that play a role in the stabilization of the protein, anchoring the kinase domain to the phosphatase domain [12]. Another mutation in PNKP at the same position (Gln517Leufs*24) has been shown to cause ataxia (including polyneuropathy) [12]. Protein modeling of PNKP with and without the Gln517ter mutation shows that this mutation is predicted to be pathogenic since the fixation of the C-terminus appears essential for stabilizing the relative domain orientation and for stabilizing the conformation of the ADP-binding site, which involves Y515 close to the site of truncation. Due to the lack of the stabilizing interactions with the other domains, the shorter carboxy-terminus of the variant is predicted to become flexible (Fig. 3c) thus leading to a distorted ADP-binding site and consequently a reduced enzymatic activity. This result in that damaged DNA, mainly by oxidative stress at the nervous system, cannot be repaired efficiently, with a subsequent transcription interference and ultimately cell death [13]. In the present study, we investigated the eldest individuals carrying mutations in PNKP to date. Contrary to what is described for AOA4 [12, 14‐17], the four reassessed individuals developed normally until the beginning of the third decade of life when gait disturbances and falls began. The disease in our patients progresses slowly, requiring a wheelchair near the sixth decade. Proximal muscle strength is preserved even in patients with the most advanced disease, and deterioration in gait was associated with postural instability rather than weakness. Cerebellar involvement occurs in all individuals, including the youngest with slurred speech-language and a wide-based gait. The two reanalyzed compound heterozygous individuals presented with oculomotor apraxia, more severe in the oldest (CMT1003) than in the youngest (CMT1190). This sign was not present in the reexamined homozygous individuals. In fact, the only finding on them related to ocular motility is the presence of slow saccadic movements in the older woman (B4.2). However, a 57-year-old male (B4.1) sibling of female B4.2 also homozygous for the p.Gln517del variant, presented with severe oculomotor apraxia and is currently wheelchair-bound. Oculomotor apraxia was not observed in the CMT2B2 individuals investigated 20 years ago.

Laboratory findings showed a small coincidence in the pattern of the biological parameters as described in other individuals that carry PNKP mutations [12, 15‐17]. Only patient CMT1190 presents hypoalbuminemia, hypercholesterolemia, and elevated IgE with normal alpha-fetoprotein as described in other patients (Table 4). The pattern of elevation or decrease of these biological parameters was not consistent in the other patients described in this study. Besides, there is no relationship between the severity of the phenotype and the levels of alpha-fetoprotein, IgE and albumin, contrary to what it has been reported in other patients with AOA4 [16, 17]. Curiously, our female patient that presents lower limb lymphedema, has no albumin alteration as described previously in a Norwegian female patient, undermining the theory that lymphedema was secondary to hypoalbuminemia [15].

The occurrence of individuals homozygous or compound heterozygous for PNKP variants which are either homozygous or heterozygous for the MED25 variant indicates that the MED25 variant is a hitchhiker variant in linkage disequilibrium on the same haplotype with the PNKP variant p.Gln517del. Despite its relatively high population frequency (1067/274,696; gnomAD All), no additional CMT2 patients have been identified with variants in MED25. Only three patients were recently reported, but these patients had additional variants in other CMT-related genes, which could well explain their phenotype [3].

In our previous study, we demonstrated that in rats Med25 expression correlated with Pmp22 dosage and expression, which is interesting because Pmp22 is a gene involved in demyelinating peripheral neuropathies. We proposed that both Med25 and Pmp22 expression are regulated by neuron-Schwann cell interactions. In addition, we demonstrated that the p.Ala335Val variant in the mediator of transcription MED25 increases the activation of the target genes in the peripheral nervous system [2]. Considering this, and that both the p.Ala335Val mutation in MED25 and a mutation in codon 517 of PNKP have shown to modify the codified protein’s function [2, 12], and the late age of onset and mild affectation of the CR-P patients, it would be possible to hypothesize that the MED25 variant confers a protective effect in them. In order to test this, patients that only present the PKNP variant would be useful to compare them clinically with patients of the CR-P family.

The involvement of PNKP in neurodegenerative disorders was already reported by Poulton et al. (2013) who presented two cases affected with a progressive polyneuropathy with early onset, severe progressive cerebellar atrophy, microcephaly, mild epilepsy, and intellectual disability. A homozygous variant (c.1250_1266dup, p.Thr424GlyfsX48) in PNKP was identified in these cases [6]. This gene, mainly related so far with AOA4 and MCSZ, has also been found through exome analysis in an individual initially diagnosed with an axonal form of Charcot-Marie-Tooth disease. This individual was homozygous for the variant p.Thr408del, also identified in our study [14]. Therefore, this study confirms the role of PNKP mutations in peripheral polyneuropathies and add data associated to the variability of the PNKP-related phenotype, due to the late age of onset of the neuropathy in our PNPK mutated patients. In conclusion, we provide evidence that PNKP is the main gene related to CMT2B2 instead of MED25, and that it should be considered as a gene involved in an axonal peripheral neuropathy with late age onset, cerebellar atrophy, and with or without oculomotor apraxia.