A Novel c.968C > T homozygous Mutation in the Polynucleotide Kinase 3′ − Phosphatase Gene Related to the Syndrome of Microcephaly, Seizures, and Developmental Delay

Abstract

Microcephaly is defined by a head circumference that is at least two standard deviations below the mean for age and sex of the general population in a specific race. Primary microcephaly may occur as an isolated inborn error, which may damage to the central nervous system or as part of the congenital abnormalities associated with genetic syndrome, affecting multiple organ systems. One of the syndromic forms consists of microcephaly, seizures, and developmental delay caused by biallelic mutations in the gene that encode polynucleotide kinase 3′ − phosphatase protein (PNKP). In this article, we reported a newborn male who presented with microcephaly, severe developmental delay, and early-onset refractories seizures, caused by a novel homozygous mutation of the PNKP gene.

Introduction

Microcephaly is usually defined by the measurement of occipital-frontal circumference (head circumference) that is more than 2 standard deviations (SDs) below the mean for age and gender in a specific race. To distinguish the causes, microcephaly is categorized as primary or secondary. ¹ Primary microcephaly (MCPH, for “microcephaly primary hereditary”) has a wide variety of causes, such as toxic exposures, in utero infections, metabolic, or genetic conditions. These incidents usually occur within the first 7 months of pregnancy. Secondary microcephaly appears when the brain, roughly normal in size at birth, does not grow thereafter. It could be resulted from brain infection, traumatic injury, or postnatal oxygen deprivation of the brain in the last 2 months of the third trimester of pregnancy. Secondary microcephaly also can occur in association with certain metabolic disorders or genetic syndromes, such as Rett syndrome. ^{1 2} Microcephaly is often congenital, meaning a baby is born with the condition, which can be classified as microcephaly vera or microcephaly with an abnormal or simplified gyral pattern. The former is characterized by intellectual disability without any structural abnormality in the brain, while the latter is associated with structural brain malformations—such as gyrification abnormalities and agenesis of the corpus callosum—along with possible perturbations of neuronal migration. Regardless of the causes or mechanisms of congenital microcephaly, seizure is often accompanying along with microcephaly and part of the clinical spectrum. ¹

When primary microcephaly is caused by gene mutations, a genetically heterogeneous condition, it could be part of a syndrome or an isolated finding. Almost all forms of microcephaly are etiologically linked to mutations in single genes that play a role in cell division, in the function of centrosomes, or in mechanisms involved in DNA damage and repairs. One of the syndromic forms of microcephaly is microcephaly, seizures, and developmental delay (MCSZ), a rare autosomal recessive neurodevelopmental disorder caused by biallelic mutations in the polynucleotide kinase 3′ − phosphatase ( PNKP ) gene. ^{1 3 4 5}

Materials and Methods

Exome sequencing was performed using extracted genomic DNA (MagnaPure, Roche) isolated from the peripheral blood of the proband and both parents. Libraries were prepared using the Ion AmpliSeq Exome Kit (Life Technologies, Thermo Fisher Scientific) and quantified by quantitative polymerase chain reaction. The enriched libraries were prepared using Ion Chef and sequenced on PI Chip in the Ion Proton System (Life Technologies) to provide >90% of amplicons covered with at least 20 × . Signal processing, base calling, alignment, and variant calling were performed on a Proton Torrent Server using the Torrent Suite Software. Variants were annotated using Ion Reporter Software. Pedigree analysis was performed using the Genetic Disease Screen trio workflow. Variant filtering and prioritization were performed with an in-house software and a local database. Candidate variants were visualized using Integrative Genomics Viewer and evaluated based on stringent assessments of both the gene and variant levels, taking into consideration both the patient's phenotype and the inheritance pattern. Variants were then classified following the guidelines of the American College of Medical Genetics and Genomics. A board-certified molecular clinical geneticist annotated each variant as pathogenic, likely pathogenic, variant of uncertain significance, or likely benign for reporting. In each case, causal variants were discussed with the referring physician and/or clinical geneticist. The identified variants were further confirmed by Sanger sequencing.

The next step was a thorough literature search and review to collect the mutations in the PNKP gene from the previously published cases. By doing that, we were able to compare the type and location of the mutations and their effects to the affected patients.

In accordance to the policies of our hospital, parental consent was obtained prior to the offering of genetic test. Permission to publish the clinical data and photos of the proband has been obtained as well. Furthermore, the Ethics Review Committee of our hospital approved this study.

Results

The patient was born, as a second child, to nonconsanguineous healthy parents whose first pregnancy was an uneventful 39-week pregnancy. During prenatal examinations, intrauterine growth retardation and microcephaly of the fetus were identified.

The patient was born at 38 ^3/7 weeks of gestation via induced vaginal delivery. The birth weight was 2,580 g (5–10th centile), length 46 cm (5–10th centile), and head circumference 30 cm (below the 3rd centile). The clinical examination at birth revealed dysmorphic craniofacial features which included: severe microcephaly, a depressed nasal bridge, a sloping forehead, and a short neck ( Fig. 1 ). Hypotonia and limitations of global movements were also observed.

Fig. 1.

A photograph of the patient showing a severe microcephaly, a depressed nasal bridge, a sloping forehead, and a short neck.

At 3 hours of age, he was admitted because of refractory seizures, which were eventually controlled by a combination of phenobarbital, levetiracetam, clobazam, and phenytoin. His capacity of oral intake was adequate until 9 months after birth. Since then, feeding problems appeared, which necessitated tubing and subsequently a percutaneous endoscopic gastrostomy at the age of 15 months.

At 11 months, the head circumference was 33 cm (<3rd centile). He produced incomprehensive sounds and he was unable to focus or follow to elevate his head when prone or to turn to the side. He was not creeping, not reaching toward objects, not recognizing his mother, and not responding to his name. He could not roll over, sit independently, or say any meaningful words. In the patient's physical examination, microcephaly, depressed nasal bridge, and short neck remained. The anterior fontanelle was closed. Neurological examination noted central hypotonia with brisk deep tendon reflexes.

The investigations, which included screening of metabolic disorders and TORCH infections (toxoplasmosis, rubella, cytomegalovirus, herpes simplex, zika, and HIV), and a chromosome microarray yielded normal results; likewise, the G-banded chromosome analysis showed a normal male karyotype (46, XY). Brainstem evoke response audiometry and ophthalmic examination were normal, and there were no any abnormal results in both abdominal and cardiac echoes. The bone scan revealed a generalized decrease in his bone density with a small cranial vault (microcephaly).

Generalized epilepsy with some degree of burst suppression was observed on the electroencephalogram. The brain magnetic resonance images (MRI) revealed microcephaly with a simplified gyral pattern and cerebellar hypoplasia. Both the white matter and corpus callosum volumes were proportionally smaller than the volume of the cerebrum, which is indicative of brain atrophy ( Fig. 2 ).

Fig. 2.

Brain magnetic resonance imaging of the patient showing brain atrophy with a simplified gyral pattern and cerebellar hypoplasia.

The exome sequencing detected a causative variant in PNKP gene, which was the only variant identified by the annotation and prioritization pipeline. The variant—located at genomic position chr19:50,365,689—was a homozygous c.968C > T mutation in exon 11 and was predicted to result in a p.Thr323Met amino acid change in the PNK protein (NM_007254.3), which generates a missense mutation in the phosphatase domain of the protein. This mutation was inherited from both patients, carriers of a heterozygous mutation. These findings were confirmed by Sanger sequencing ( Fig. 3 ). This variant was reported in the Exome Aggregation Consortium database with an allele frequency lower than 0.02%. The mutation was classed as “probably damaging” by SIFT, PROVEAN, PolyPhen-2, MutationTaster, Mutation Assessor, LRT, CONDEL, and MetaSVM. It is predicted to produce a decrease in the stability of the PNK protein.

Fig. 3.

Image of the sequencing chromatograph that shows the variant found in the patient (highlighted in blue).

Discussion

The maintenance of DNA integrity is crucial for all kind of cells, especially neurons that are extremely sensitive to mutations altering DNA repairs. DNA damages are a constant threat to cells, which can occur spontaneously via DNA replication or transcription, or endogenously produced by reactive oxygen species generated in oxidative metabolism. In fact, the most frequent DNA damage in cells comes from the action of reactive oxygen species that results in DNA single-strand breaks. ⁵ Congenital defects in single-strand break repair mechanisms affect almost exclusively the nervous system, leading to neurodegeneration. High level of vulnerability can be explained by the high rate of oxygen consumption of neural cells. ^{5 6} Furthermore, other kinds of DNA damage have an influence on the nervous system as well like unrepaired DNA double-strand breaks—which can result in abnormalities in the development of the brain, and microcephaly was one of the possible outcomes caused by these mutations, especially in the neurogenesis process. ^{7 8} The different types of mutations causing unrepaired DNA damage can lead to diverse alterations, including developmental defects, degenerative disease, aging, or cancer. ⁹ In particular, DNA repair malfunctions can accelerate the cell death process, resulting in underdevelopment of the brain and/or neurodegeneration. ¹⁰

Polynucleotide kinase 3′-phosphatase protein is an important dual-function enzyme among the various pathways of DNA damage repair ^{4 10} It consists of a C-terminal catalytic domain, where a fused bimodal phosphatase and kinase domain are located, with a forkhead-associated domain (FHA) at its N-terminus ⁹ This protein has a polynucleotide 3′-phosphatase and a polynucleotide 5′-hydroxyl kinase domain, and it has an important role in the repair of both single-strand and double-strand breaks because that its phosphatase domain removes 3′ phosphates and the kinase domain phosphorylates 5′-hydroxyl groups, which is required for DNA ligation. ^{5 11 12} The forkhead domain of PNKP interacts with either the XRCC1 or XRCC4 scaffold proteins, which are also involved in the repair of single- or double-strand break components, respectively. The most common type of endogenous DNA damage is single-strand breaks, where PNKP plays a significant role in most of the steps for repairing these breaks because of the abundance of 3′-P termini. ^{7 9 13}

A certain number of genetic syndromes have been linked to mutations in the PNKP : microcephaly with seizures (MCSZ), microcephaly associated with neurodegeneration and polyneuropathy, and ataxia with oculomotor apraxia type 4 (AOA4). ^{4 5 9 14} Both MCSZ and AOA4 are disorders characterized by a specific involvement of nervous system, but not affecting other organs and systems.

The MCSZ ( OMIM613402, also named epileptic encephalopathy, early infantile, 10) is an autosomal recessive disorder linked to a homozygous or compound heterozygous mutation in PNKP at 19q13. It is characterized by microcephaly, intractable seizures, developmental delay, and behavior problems, particularly hyperactivity. The onset of this neurodevelopmental disease is during infancy. ^{4 5 9} The clinical presentation has a wide spectrum of severity. The classical form consists of a severe early-onset infantile epileptic encephalopathy, without evidence of brain atrophy. On the other hand, there are other clinical forms with a controllable seizure and a prolonged course along with cerebellar atrophy and peripheral neuropathy. ^{4 5 15} Our patient had a severe seizure and revealed an abnormal and simplified gyral pattern ( Tables 1 and 2 ).

Table 1. Description of the cases found in the bibliographic research with mutations in polynucleotide kinase 3′ − phosphatase gene and a microcephaly, seizures, and developmental delay phenotype.

Patient	Family	Consanguinity	Mutation	Exon	c.DNA	PNKP mutation	Domains	Clinical phenotype	Neurologic findings
Shen	X3 Palestinian patients (homozygous)	Yes	Substitution	Exon 11	c.975 G > A	Glu326Lys	Phosphatase	Microcephaly Seizures Developmental delay	White matter defects
Shen	X1 Turkish patient (homozygous)	No	Duplication	Exon 14	c.1250_1266dup	Thr424GlyfsX48	Kinase	Microcephaly Seizures Developmental delay	White matter defects
Shen	X1 Saudi Arabia patient (homozygous)	No	Duplication	Exon 14	c.1250_1266dup	Thr424GlyfsX48	Kinase	Microcephaly Seizures Developmental delay	White matter defects
Shen	X1 European patient (2heterozygous)	No	Duplication substitution	Exon 14 Exon 5	c.1250_1266dup c.526 C > T	Thr424GlyfsX48 Leu176Phe	Kinase Phosphatase	Microcephaly Seizures Developmental delay	White matter defects
Shen	X1 European patient (2heterozygous)	No	Duplication Deletion	Exon 14 Del intron 15	c.1250_1266dup g.5646_5662del	Thr424GlyfsX48 mRNA splicing	Kinase	Microcephaly Seizures Developmental delay	White matter defects
Poulton	X1 Dutch patient (homozygous)	Yes	Duplication	Exon 14	c.1250_1266dup	Thr424GlyfsX48	Kinase	Microcephaly Seizures Ataxia Neurodegeneration	Cerebellar atrophy
Nakashima	X1 Japanese patient (2heterozygous)	No	Substitution Substitution	Exon 2 Exon 8	c.163G > T c.874G > A	Ala55Ser Gly292Arg	FHA Phosphatase	Microcephaly Seizures Developmental delay	White matter defects Hearing loss
Carvill	Single individual (homozygous)	Not available	Substitution	Exon 1	c.58 G > A	Pro20Ser	FHA	Not microcephaly Seizures Not developmental delay	Not available
Nair	X1 Emirati patient (homozygous)	Yes	Substitution	Exon 15	c.1385 G > C	Arg462Pro	Kinase	Microcephaly Severe seizures Developmental delay Primordial dwarfism	Agenesis of the corpus callosum Cerebellar atrophy
Taniguchi-Ikeda	X1 Japanese (2heterozygous)	Not available	Substitution Deletion	Exon 11 Exon 15	c.1028C > T c.1313_1318del	Pro343Leu Ala438_Arg439del	Kinase Kinase	Microcephaly Severe seizures Developmental delay	Cerebellar atrophy Pachygyria Hypoplastic brainstem
Taniguchi-Ikeda	X1 Japanese (homozygous)	Yes	Substitution	Exon 15	c.1299–2A > G	Skipping exon 15	Kinase	Microcephaly Severe seizures Developmental delay Oculomotor apraxia	Cerebellar atrophy Spinocerebellar degeneration
Entezam	X1 Iranian (homozygous)	Yes	Substitution	Exon 13	c.1133A > C	Lys378Thr	Kinase	Microcephaly Severe seizures Developmental delay Oculomotor apraxia Neuropathy	Cerebellar atrophy
Gatti	X1 Italian patient (homozygous)	No	Duplication	Exon 14	c.1253_1269dup	Thr424Glyfs49	Kinase	Microcephaly Severe seizures Developmental delay Oculomotor apraxia Neuropathy	Cerebellar atrophy
Gatti	X1 Italian patient (homozygous)	No	Duplication	Exon 14	c.1274_1284dup	Ala429Thrfs42	Kinase	Microcephaly Not seizures Developmental delay Oculomotor apraxia Neuropathy	Cerebellar atrophy
Bitarafan	X1 Iranian patient (heterozygous)	Yes	Deletion Duplication	Exon 14 Intron 14	c. 1298_1269del c.1253_1269dup	- Thr424Glyfs49	Kinase Kinase	Microcephaly Severe seizures Developmental delay Ataxia	Callosal dysgenesis
Our case	X1 Spanish patient (homozygous)	No	Substitution	Exon 11	c. 968C > T	p.Thr323Met	Phosphatase	Microcephaly Seizures Developmental delay	Cerebral atrophy Cerebellar hypoplasia

Abbreviations: FHA, forkhead-associated domain; PNKP, polynucleotide kinase 3′ − phosphatase.

Table 2. Description of the cases found in the bibliographic research with mutations in polynucleotide kinase 3′ − phosphatase gene and an ataxia with oculomotor apraxia type 4 phenotype.

Patient	Family	Consanguinity	Mutation	Exon	cDNA	PNKP mutation	Domains	Clinical phenotype	Neurologic findings
Bras	X2 Portuguese patients (heterozygous)	No	Substitution Duplication	Exon 12 Exon 14	c.1123 G > T 1253_1269dup	Gly375Trp Thr424Glyfs49	Kinase	Dystonia Oculomotor apraxia Neuropathy Cognitive impairment	Cerebellar atrophy Brainstem atrophy
Bras	X1 Portuguese patient (homozygous)	Yes	Substitution	Exon 12	c.1123 G > T	Gly375Trp	Kinase	Dystonia Oculomotor apraxia Neuropathy Cognitive impairment	Cerebellar atrophy
Bras	X1 Portuguese patient (heterozygous)	No	Deletion Insertion	Exon 13 Exon 15	c.1221_1223del c.1549_1550ins	Thr408del Gln517Leufs24	Kinase Kinase	Dystonia Oculomotor apraxia Neuropathy Ataxia Cognitive impairment	Cerebellar atrophy
Bras	X1 Portuguese patient (heterozygous)	No	Deletion Insertion	Exon 13 Exon 14	c.1221_1223del c.1315_1329ins	Thr408del Arg439Glyfs51	Kinase Kinase	Dystonia Oculomotor apraxia Neuropathy	Cerebellar atrophy
Bras	X1 Portuguese patient (homozygous)	Yes	Substitution	Exon 12	c.1123 G > T	Gly375Trp	Kinase	Dystonia Oculomotor apraxia Neuropathy Ataxia	Cerebellar atrophy
Bras	X1 Portuguese patient (heterozygous)	Yes	Substitution Insertion	Exon 12 Exon 14	c.1123 G > T c.1322_1323ins	Gly375Trp Gly442Alafs27	Kinase Kinase	Oculomotor apraxia Neuropathy Ataxia	Cerebellar atrophy
Bras	X2 Portuguese patients (homozygous)	Yes	Substitution	Exon 12	c.1123 G > T	Gly375Trp	Kinase	Oculomotor apraxia Neuropathy	Cerebellar atrophy
Bras	X2 Portuguese patients (homozygous)	No	Substitution	Exon 12	c.1123 G > T	Gly375Trp	Kinase	Dystonia Oculomotor apraxia Neuropathy Cognitive impairment	Cerebellar atrophy Brainstem atrophy
Paucar	X1 Swedish patient (heterozygous)	No	Substitution Substitution	Exon 14 Exon 15	c.1196T > C c.1385G > C	Leu399Pro Arg462Pro	Kinase Kinase	Neuropathy Ataxia Cognitive impairment	Cerebellar atrophy
Tzoulis	X1 Norwegian patient (heterozygous)	No	Substitution Deletion	Exon 14 Exon 16	c.1196T > C c.1393_1396del	Leu399Pro Glu465ter	Kinase Kinase	Oculomotor apraxia Neuropathy Ataxia Cognitive impairment	Cerebellar atrophy
Scholz	X1 German patient (heterozygous)	No	Duplication Substitution	Exon 14 Exon 17	c.1253_1269dup c.1545C > G	Thr424fs*49 Tyr515*	Kinase Kinase	Neuropathy Ataxia Cognitive impairment	Cerebellar atrophy
Schiess	X1 patient (homozygous)	Not available	Deletion	Exon 14	c.1189_1191del18	(splicing)	Kinase	Oculomotor apraxia Neuropathy Ataxia Cognitive impairment	Cerebellar atrophy
Rudenskaya	X1 Byelorussian patient (heterozygous)	No	Substitution Duplication	Exon 12 Exon 14	c.1123G > T c.1270_1283dup	Gly375Trp Ala429fs	Kinase Kinase	Oculomotor apraxia Neuropathy Ataxia Cognitive impairment	Cerebellar atrophy

Abbreviation: PNKP, polynucleotide kinase 3′ − phosphatase.

There were no any clinical reports of a higher frequency of infections in patients with MCSZ patients. Therefore, it seems that MCSZ is not linked to immunodeficiencies. ^{5 9 10} As microcephaly is linked with defects in repairing double-strand breaks, they must be implicit in MCSZ. On the other hand, the lack of neurodegenerative changes suggests that problems in the repair of single-strand breaks are not relevant in this pathology, although PNKP participates in both DNA repair pathways. ⁹

The AOA4 (OMIM616267) is an autosomal recessive neurological disorder. A typical feature of this disorder is the onset of dystonia and ataxia in the first decade of life. Other manifestations are oculomotor apraxia (with abnormal saccadic eye movements), cerebellar degeneration, sensorimotor axonal neuropathy, and extrapyramidal features. Meanwhile, cognitive impairment can be found in some patients. ^{9 14} The course of this disorder is progressive and by the second or third decades of life most patients rely on a wheelchair to move around. ¹⁴

As previously mentioned, PNKP is a modular protein with three domains: an amino-terminal FHA domain, a DNA phosphatase domain, and a DNA kinase domain. ¹⁶ These three domains have been affected by different mutations in the MCSZ ( Figs. 4 and 5 ). The pathogenic effect was more frequently related to the mutations that affected exons 11 to 15, and mostly to 14 for MCSZ and 12 for AOA4. The different phenotypes so far associated with mutations in PNKP do not seem to correlate with the location of the mutation. ¹⁴ The same homozygous variant, p.Thr424Glyfs*49 (c.1253_1269dup) located in the kinase domain of the protein, has been found in individuals with MCSZ, ⁵ and in individuals with neurodegenerative disorder AOA4. ¹⁰ For AOA4, Bras et al found the same PNKP mutation, c.1123G > T (p.Gly375Trp), in three of the studied families; this mutation was later described in another patient. ¹⁷ Variants found to be associated with AOA4 were all located in or adjacent to the kinase domain of the protein. Mutations in the phosphatase or FHA domain have not been found in patients with AOA4. ^{14 16 17}

Fig. 4.

A representation of chromosome 19 with an arrow showing the location of PNKP gene within the chromosome. Graph from condition, gene, or chromosome summary: National Library of Medicine (US). Genetics Home Reference [Internet]. Bethesda (MD): The Library; 2019 July 16. PNKP gene; [reviewed 2018 June; cited 2019 August 06]; [∼6 screens]. Available at: https://ghr.nlm.nih.gov/gene/PNKP . PNKP, polynucleotide kinase 3′ − phosphatase.

Fig. 5.

Location and number of mutations in PNKP gene (number of mutations in each exon of PNKP gene in black outlined squares). The figure shows that mutations are located mainly between exons 11 and 15, conditioned by the recurring mutations of c.1123 G > T in exon 12 and 1250_1266dup in exon 14. Adapted from Atlas of Genetics and Cytogenetics in Oncology and Haematology. Available at: http://AtlasGeneticsOncology.org . PNKP, polynucleotide kinase 3′ − phosphatase.

The severity of the phenotype may be better linked to the type of mutations. Shen et al found that a splicing mutation is associated with more moderate symptoms and suggested a single common homozygous region on chromosome 19q in exon 14 (1250_1266dup c.526 C > T) for four of his five families, which is also repeated in the family described by Poulton et al. ^{5 10} Other regions for MCSZ have also been described, affecting exons 11 (five and our case) and 15. ^{3 5}

Mainly, alterations in the mechanisms of repairing double-strand breaks result in microcephaly, while mutations which compromise the integrity of single-strand break repairing processes are related to neurodegeneration. MCSZ and AOA4 are easily distinguishable from each other: the phenotype of MCSZ is similar to that found in other disorders in which mechanisms of double-strand break repairing are altered, whereas defects in the repair of single-strand breaks are linked to AOA4. ^{4 5 9 14}

The not previously described PNKP missense mutation identified in our patient on exon 11 results in a p.Thr323Met change in the protein. This is a highly conserved residue that lies in the phosphatase domain. The same mutation was heterozygous in both unaffected parents. Mutations in this domain have been only described previously in three patients and they all had MCSZ phenotype. ¹⁸

In conclusion, we identified a mutation in the PKNP gene which results in a phenotype similar to those described in other reported cases of MCSZ, with evidences of brain anomalies like cerebral atrophy and severe seizures, except for the absence of behavioral changes related to hyperactivity.