Microcephaly, seizures and developmental delay caused by two novel mutations in the PNKP gene: a case report

Introduction

Microcephaly is characterized by an occipitofrontal head circumference (OFC) that is two standard deviations (SDs) below the mean expected for a given age, gender and population. The prevalence of this condition varies significantly, with estimates ranging from 1.3 to 150 cases per 100,000 live births (1). It is a chronic condition and prognosis is largely contingent upon the underlying cause and the severity. It may also be linked to various complications, which include intellectual disabilities, developmental delays, epilepsy and cerebral palsy (1).

Cerebral growth is reliant on normal neuronal tissue proliferation, which necessitates continuous cellular division. Numerous genetic neurodevelopmental and neurodegenerative disorders have been identified as resulting from mutations in DNA repair genes. This underscores the critical role of these pathways in neurogenesis (2).

Microcephaly, seizures and developmental delay (MCSZ; OMIM #613402) is a rare autosomal recessive neurodevelopmental disorder that is associated with a bifunctional enzyme known as polynucleotide kinase 3’-phosphatase (PNKP) (3,4). This enzyme functions as both a DNA 3’-phosphatase and a DNA 5’-kinase. The gene is located on chromosome 19q13.33, encodes an essential enzyme involved in DNA repair processes and is particularly vital for the proper functioning of the nervous system (4,5).

Here, we report compound heterozygote mutations in the PNKP gene in a 4-month-old boy with non-consanguineous parents. The mutations found were c.1283_1287dup and c.1299-1G>A. Our study expands the range of known variants in the PNKP gene and improves the understanding of clinical features, genetic characteristics and diagnostic protocols of MCSZ. We present this article in accordance with the CARE reporting checklist (available at https://tp.amegroups.com/article/view/10.21037/tp-2025-1-820/rc).

Case presentation

Patient and clinical details

The proband was a male child born to non-consanguineous Chinese parents (Figure 1). He was a baby conceived via assisted reproductive technology (in vitro fertilization, IVF) (vaginal delivery at 38+5 weeks, G2P1, no asphyxia and birth weight of 3,000 g). Microcephaly and oligohydramnios were detected at 32 weeks of gestation, leading to the mother’s hospitalization. After birth, he was hospitalized for jaundice in a local hospital for 1 week. Neither the proband’s parents nor grandparents showed any clinical symptoms. The proband developed recurrent convulsions at the age of 4 months, without fever, vomiting or diarrhea. The proband presented with tonic-clonic seizures. After the second convulsion, the proband was admitted to the Children’s Hospital of Soochow University for further treatment.

Figure 1 Pedigree of the Chinese family with MCSZ. Squares and circles denote males and females, respectively. Roman numerals indicate generations. An arrow indicates the proband (II-1). MCSZ, microcephaly, seizures and developmental delay.

At the age of 4 months, the head circumference of the proband was 35 cm, increasing to 39 cm at the age of 9 months. He showed delays in motor development. He was able to raise his head steadily at the age of 4 months, sit unaided at the age of 9 months, but had difficulty standing and walking independently. At the age of 5 years old, he had not learned to speak and was unable to stand unaided. Physical examination revealed muscle strength at level 4, lower muscle tone than his peers, preserved physiological reflexes and negative pathological signs. His cardiovascular, abdominal, urogenital, electrolyte, liver and kidney functions were normal.

The proband’s levels of serum ammonia and lactic acid were found to be marginally increased. However, findings from the blood tandem mass spectrometry analysis remained within the normal range. The chromosomal karyotyping of the proband was normal.

Intracranial magnetic resonance imaging (MRI) indicated a closed anterior fontanelle and insufficient brain volume without any apparent structural abnormalities at 4 months of age (Figure 2A). At the age of 17 months, a cranial computed tomography (CT) scan identified multiple anomalies, which included craniosynostosis, bilateral widening of the lateral ventricles and bilateral frontotemporal cortical atrophy, more pronounced in the frontal lobes (Figure 2B).

Figure 2 Clinical data of the proband with MCSZ. (A) MRI at 4 months showed a closed anterior fontanelle and insufficient brain volume, with no significant structural abnormalities detected. The red arrows indicate widened extracerebral space. (B) At 17 months of age, head CT demonstrated bilateral frontotemporal cortical atrophy, more pronounced in the frontal lobes, along with bilateral widening of the lateral ventricles. The red arrows indicate the widened bilateral lateral ventricles. (C,D) Video electroencephalogram examination results of the proband (C: waking period, D: sleep period). (E,F) Sequence chromatograms of the compound heterozygous mutations. Mutations are indicated by red arrows. CT, computed tomography; MCSZ, microcephaly, seizures and developmental delay; MRI, magnetic resonance imaging.

The video electroencephalogram (EEG) (Figure 2C,2D) indicated abnormalities, though no epileptic seizures were captured during monitoring. During both wakefulness and sleep, medium- to high-amplitude slow waves intermixed with multifocal spikes and sharp waves were observed over both cerebral hemispheres. These discharges were bilateral, asymmetric and asynchronous. During sleep, bilateral occipital regions showed bursts of medium- to high-amplitude sharp and slow wave complexes at 2–2.5 Hz.

Throughout the follow-up period (over 4 years), the proband exhibited slow weight gain, weighing only 12.5 kg at the age of 5 years, while symptoms of microcephaly progressively worsened. He was treated with four anti-seizure medications (ASMs), namely levetiracetam, topiramate, nitrazepam, and perampanel, but did not achieve satisfactory control. Prior to 3 years of age, he was hospitalized multiple times due to seizures, which occurred 2 to 3 times per month. Although the ketogenic diet was recommended by the physicians, his parents declined this treatment option. After the age of 3 years, the frequency of seizures decreased to 2 or 3 episodes per year.

The neuropsychological development test for children aged 0–6 years was conducted when the child was 1 year and 10 months old. It indicated significant developmental delays that may require further intervention (Table 1). All procedures performed in this study were in accordance with the Declaration of Helsinki and its subsequent amendments. This study was approved by the Ethics Committee of Children’s Hospital of Soochow University (No. 2022CS051). Written informed consent was obtained from the patient’s guardians for the publication of this case report and accompanying images. A copy of the written consent is available for review by the editorial office of this journal.

Identification and analysis of PNKP variants

Novel compound heterozygous variants c.1283_1287dup (p.S430Pfs*39) and c.1299-1G>A were identified in the PNKP gene (NM_007254) of the proband using whole exome sequencing (WES). Sanger sequencing confirmed that the variants were novel. The c.1283_1287dup (p.S430Pfs*39) variant was inherited from his father (Figure 2E), whilst c.1299-1G>A was inherited from his mother (Figure 2F).

The premature termination codon of c.1283_1287dup (p.S430Pfs*39) is located within the last 50 3' nucleotides of the penultimate exon. NMD is not expected to occur (PVS1_Strong). This variant has not been documented in prior studies and is not found in the gnomAD, 1000 Genomes or ExAC databases (PM2). The variant was confirmed to be in trans with pathogenic variant c.1299-1G>A in the proband (PM3). The alignment of PNKP protein sequences across various species demonstrates a high degree of conservation at the S430 amino acid position, which suggests that it may be integral to the stability and functionality of the protein (Figure 3A). In accordance with the guidelines set forth by the American College of Medical Genetics and Genomics (ACMG) for the evaluation of sequence variants, the variant c.1283_1287dup (p.S430Pfs*39) was categorized as “Likely Pathogenic”.

Figure 3 The alignment of PNKP protein sequences across various species and structural alteration of the PNKP protein. (A) Conservation of the S430Pfs*39 variant across various species. (B,C) Amino acid and conformation changes in the S430Pfs*39 polypeptide. (B) is the wild-type and (C) is the mutant.

Loss of function of PNKP is a well-established mechanism (6). The variant c.1299-1G>A was identified as a canonical splice site (PVS1), has not been reported in previous literature and is not found in the 1000G or ExAC databases. It is also extremely rare in the gnomAD database (PM2). The variant was confirmed to be in trans with likely pathogenic variant c.1283_1287dup (p.S430Pfs*39) in the proband (PM3). In accordance with the ACMG guidelines, c.1299-1G>A was classified as “Pathogenic”.

Structural alteration of the PNKP protein

Three-dimensional protein structure prediction showed that the S430Pfs*39 variant results in shorter protein length and destroys the AAA_33 domain, which may affect protein spatial conformation, protein stability and enzyme activity (Figure 3B,3C).

RNA-seq analysis revealed exon 15 skipping and intron 14 retention in the PNKP gene of the proband

RNA sequencing (RNA-seq) was used to explore the transcriptional modifications of PNKP, and to identify de novo splice sites. The RNA-seq analysis validated the presence of the c.1299-1G>A variant and indicated that the proband harbored two abnormal transcripts. Transcript A was characterized by the skipping of exon 15, while Transcript B involved the retention of intron 14 (Figure 4A,4B). Analysis of Transcript A revealed a novel splicing pattern, which led to a frameshift and the generation of a premature termination codon (c.1299_1387del88bp, p.Arg433Serfs*5). Retention of intron 14 in Transcript B was predicted to result in a frameshift, along with a premature termination codon (c.1298_1299ins76bp, p.Y434*). The RNA sequencing results confirmed that the variant had an impact on mRNA splicing.

Figure 4 RNA-seq results for the proband and schematic presentation of the linear PNKP protein with all variants. (A,B) The red dotted line points to the mutation site. The proband has two abnormal transcripts, which present as exon 15 skipping and intron 14 retention. (C) The red font indicates reported variants in this study. Green indicates the FHA domain at the N-terminus, red indicates the DNA phosphatase domain and blue indicates the C-terminal DNA kinase domain. FHA, fork-head associated.

Discussion

In this investigation, we documented a two-generation family in China that exhibited an autosomal recessive neurodevelopmental disorder, which is distinguished by microcephaly, seizure activity and delays in development. WES was used to identify two novel heterozygous variants in the PNKP gene: c.1299-1G>A and c.1283_1287dup (p.S430Pfs*39). The clinical features of the proband were consistent with those found in previous studies (5). An RNA-seq analysis demonstrated that the splicing variants created two abnormal transcripts. These transcripts were predicted to interrupt the translational process, resulting in truncated proteins. It was assumed that the variants were the genetic cause of the disorder in this family. Our study not only adds two new pathogenic variants to the mutational spectrum of the PNKP gene but also lays a foundation for future studies on the correlation between genotype and phenotype.

The MCSZ disorder is a rare neurodevelopmental disorder characterized by microcephaly. Epidemiological data regarding the incidence of MCSZ remains unreported. Clinical features include microcephaly, early-onset refractory epilepsy, developmental delays and behavioral problems, such as attention deficit hyperactivity disorder (ADHD). The limited numbers of patients documented thus far show a significant level of phenotypic variability (Table 2), with a spectrum that varies from a traditional manifestation characterized by the absence of brain atrophy to clinical deterioration and a neurodegenerative presentation. The latter is characterized by the presence of progressive cerebellar atrophy, alongside sensorimotor peripheral neuropathy (5,15). The epileptic phenotype also demonstrates considerable variability. Individuals diagnosed with the classical variant of the disorder have been reported to suffer from severe early-onset infantile epileptic encephalopathy. Conversely, patients with the progressive neurodegenerative variant display a less severe epileptic profile and experience sporadic seizures that generally show improvement over time. It was not until 2010 that a mutation in the PNKP gene, found in six unrelated families, was recognized as the genetic cause of MCSZ (5).

The PNKP gene is found on chromosome 19q13.33 and consists of 17 exons that encode a 521 amino acid peptide. This gene encodes polynucleotide kinase-phosphatase. The enzyme exhibits dual functionality as a DNA 3’-phosphatase and a DNA 5’-kinase, making it essential for the repair processes of both single-stranded and double-stranded breaks in DNA (6,7). The PNKP protein has three distinct domains: an N-terminal fork-head associated (FHA) domain, which is crucial for localization of the protein to DNA repair sites, a DNA phosphatase domain and a C-terminal DNA kinase domain (16). Collectively, these domains form the catalytic region that encompasses amino acid residues 166 to 328 and 399 to 489 (3).

Besides MCSZ, alterations in the PNKP gene have been identified as causative factors for two uncommon neurological disorders: the autosomal recessive neurodegenerative condition Ataxia-ocular motor apraxia type 4 (AOA4) and the inherited peripheral neuropathy Charcot-Marie-Tooth disease type 2B2 (CMT2B2) (8). Research has indicated that the nature of DNA breaks, whether single-stranded or double-stranded, along with the timing of neuronal cell injury, may play a pivotal role in shaping the phenotype. The condition known as MCSZ arises from impaired repair of double-stranded DNA breaks by PNKP, which occurs in mitotic neuronal progenitors during the developmental phase. In contrast, within postmitotic cells, PNKP is believed to significantly contribute to the repair of single-stranded DNA breaks and the maintenance of mitochondrial homeostasis. These functions may play a pivotal role in the development of late-onset and progressive neurodegenerative disorders, such as AOA4 and CMT2B2. In addition, it is conceivable that polymorphisms or variations in other genes may function as genetic modifiers, thereby exerting a functional influence on the activities of the PNKP protein and subsequently affecting the phenotypic manifestation of the disease (17,18).

To date, 49 variants of the PNKP gene have been cataloged in the Human Gene Mutation Database (HGMD) (Figure 4C) and mutations affecting PNKP have been identified across all three functional domains (18). While the precise mechanisms that create the phenotypic diversity observed in syndromes linked to PNKP variants remain elusive, it has been proposed that the most severe phenotypes may arise from significant loss of protein functionality. In contrast, milder phenotypes may be attributed to residual activity of the PNKP protein (17,18). This proposition is corroborated by findings in mice, in which complete loss of PNKP leads to nonviable embryos. However, a hypomorphic allele that reduces enzyme expression permits viability, albeit with repercussions for genome stability (19). Both mutations identified in our proband are protein truncating mutations and may result in a major loss of protein function. This explains the early age of onset and disease course of the proband.

All mutations implicated in AOA4 are localized within the kinase domain (20). This investigation pinpointed two specific mutations, c.1299-1G>A and c.1283_1287dup (p. S430Pfs*39), which also impact the DNA kinase domain. Research conducted by Taniguchi-Ikeda et al. in 2017 revealed that while pathogenic variants within the PNKP gene result in the distinct manifestations of MCSZ and AOA disorders, mutations situated in the kinase domain may exhibit a broad range of overlapping phenotypic characteristics associated with both conditions (9).

The MCSZ disorder is an exceedingly uncommon autosomal recessive genetic disorder. Despite the critical role of PNKP in DNA repair, only two cases have been documented that correlate PNKP mutations with cancer in MCSZ patients. Jiang (8) discussed the biochemical implications of two germ-line point mutations found in the PNKP gene of a 3-year-old male diagnosed with MSCZ, who presented with a high-grade brain tumor in the cerebellum (glioblastoma multiforme). Functional and biochemical analyses indicated that these PNKP mutations markedly diminish DNA kinase/phosphatase activity and modify its cellular distribution. This results in defects in the repair of single/double-stranded DNA breaks and an increased likelihood of carcinogenic transformation. In addition, Bayram reported a separate case of a child with MSCZ who subsequently developed acute myeloid leukemia (21). This further contributes to the accumulating evidence that associates PNKP with oncogenesis.

At present, MCSZ is recognized as an untreatable condition that lacks viable long-term therapeutic options. The prevailing approach involves symptomatic management aimed at mitigating seizure activity. However, this strategy frequently proves to be inadequate. Furthermore, there is a lack of definitive evidence to categorize the seizure manifestations observed in individuals with MCSZ.

Both parents often carry the disease-causing mutation and each time they have a child, there is a 25% likelihood that the child will have the disease. Other relatives of the patient’s parents may also carry the same pathogenic mutation. Therefore, when suspected cases are identified in clinical practice, the relevant genetic testing should be conducted to confirm a diagnosis. Genetic counseling is crucial for families with a history of MCSZ. It is recommended that amniocentesis is conducted between the 15^th and 23^rd weeks of any subsequent pregnancy for the purpose of prenatal diagnosis. This approach aims to mitigate the risk of recurrent MCSZ and enhance the quality of live births.

Conclusions

In summary, this is the third report of MCSZ in a non-consanguineous Chinese family. Using clinical features and biochemical markers, we hypothesized that two newly identified pathogenic variants in the PNKP gene are responsible for the disease manifestation in this patient. Our results, together with earlier research, offer substantial evidence to support the clinical variability associated with PNKP mutations, which complicates clinical diagnosis. This underscores the critical role of genetic testing in elucidating the underlying causes of such disorders. In addition, next-generation sequencing (NGS) and Sanger sequencing have been used to establish a reliable foundation for both clinical and prenatal diagnostics within this family, while also expanding the mutation spectrum associated with the PNKP gene.

Acknowledgments

The authors thank International Science Editing (http://www.internationalscienceediting.com) for editing this manuscript. The authors also thank the patient and his family for participating in this study.

Footnote

Reporting Checklist: The authors have completed the CARE reporting checklist. Available at https://tp.amegroups.com/article/view/10.21037/tp-2025-1-820/rc

Peer Review File: Available at https://tp.amegroups.com/article/view/10.21037/tp-2025-1-820/prf

Funding: This work was supported by the National Natural Science Foundation of China (No. 82171441), the National Natural Science Foundation of Jiangsu Province (No. BK20201175), the Epilepsy Research Foundation of China Association Against Epilepsy (No. CW-2022-024), the Project of Science and Technology Development Plan in Suzhou (Technology of People’s Livelihood) (No. SYSD2020122), and the Project of Youth Science and Technology in Suzhou (Science, Education and Health) (No. KJXW2022018).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tp.amegroups.com/article/view/10.21037/tp-2025-1-820/coif). The authors sincerely thank the Berry Gnomics Co., especially Xiaotian Su for the technical support. The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All procedures performed in this study were in accordance with the Declaration of Helsinki and its subsequent amendments. This study was approved by the Ethics Committee of Children’s Hospital of Soochow University (No. 2022CS051). Written informed consent was obtained from the patient’s guardians for the publication of this case report and accompanying images. A copy of the written consent is available for review by the editorial office of this journal.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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