Newly discovered variants in unexplained neonatal encephalopathy

Abstract

Background

The genetic background of neonatal encephalopathy (NE) is complicated and early diagnosis is beneficial to optimizing therapeutic strategy for patients.

Methods

NE Patients with unclear etiology received regular clinical tests including ammonia test, metabolic screening test, amplitude‐integrated electroencephalographic (aEEG) monitoring, brain Magnetic Resonance Imaging (MRI) scanning, and genetic test. The protein structure change was predicted using Dynamut2 and RoseTTAFold.

Results

15 out of a total of 113 NE Patients were detected with newly reported pathogenic variants. In this sub‐cohort, (1) seizure was the primary initial symptoms; (2) four patients had abnormal metabolic screening results, and two of them were also diagnosed with excessive blood ammonia concentration; (3) the brain MRI results were irregular in three infants and the brain waves were of moderate–severe abnormality in about a half of the patients. The novel pathogenic variants discovered in this study belonged to 12 genes, and seven of them were predicted to introduce a premature translation termination. In‐silicon predictions showed that four variants were destructive to the protein structure of KCNQ2.

Conclusion

Our study expands the mutation spectrum of genes associated with NE and introduces new evidence for molecular diagnosis in this newborn illness.

This study reports 15 novel pathogenic variants related to neonatal encephalopathy (NE). Our findings expands the mutation spectrum of genes associated with NE and introduces new evidence for molecular diagnosis in this newborn illness.

1. INTRODUCTION

Occurring in the earliest days of life in a newborn baby, neonatal encephalopathy (NE) is a syndrome characterized by disturbed neurological function including subnormal level of consciousness or seizures, often accompanied by decreased response to stimuli, weak tone, muscle hypotonia and dyspnea (No authors, 2014; Volpe, 2012). The incidence of NE is 1.6‰ for high‐income countries and 12.1‰ for low‐income (Lee et al., 2013). Neonates with moderate to severe encephalopathy have risks of death or developing neurological sequelae such as irreversible neurodevelopmental delay (Pappas et al., 2015). The currently known etiological spectrum of this difficult disease includes hypoxic‐ischemic encephalopathy, intrauterine infections, placental abnormalities, thrombophilia, coagulation defects, metabolic disorders, ischemic stroke, intracranial hemorrhage, etc. (Aslam et al., 2019; Glass, 2018; Hankins & Speer, 2003). However, in clinical practice, the cause of encephalopathy is unexplained in a considerable portion of ailing term infants (Bruun et al., 2018; Yang et al., 2020).

After the first successful application of the whole exome sequencing (WES) in capturing the causal gene of a Mendelian disease (Ng et al., 2010), the next‐generation sequencing (NGS) technologies are widely adopted to scan the flaws of individual genetic blueprint in the molecular diagnosis of diseases induced by congenital factors (Zhang et al., 2014). To investigate the underlying genetic defects of NE cases without identifiable etiological factor, Bruun et al. conduct a prospective cohort study with the utilization of WES. The research team pinpointed pathogenic variants in several known genes causing neonatal epileptic encephalopathy and reported that the genetic causes of 36% of the ailing babies were confirmed (Bruun et al., 2018). This study suggested that WES was helpful at arriving molecular diagnoses in unexplained NE.

Numerous NE‐related genes had been identified and Yang et al. classified these genes into four groups including epileptic, metabolic, mitochondrial, and syndromic‐related gene group (Yang et al., 2020). In their research, the most frequently affected gene was a seizure‐related gene named KCNQ2 (OMIM accession number: 602235). Epileptic encephalopathy (EE) accounted for the largest proportion in NE caused by a known genetic factor (Nappi et al., 2020) and the deleterious KCNQ2 variant played an essential role in the etiology of this disease subtype (McTague et al., 2016). The KCNQ2 gene maps to chromosome 20q13.33 and consists of 17 exons according to the information from the National Center for Biotechnology Information (NCBI). KCNQ2 is expressed predominantly in neurons (Gunthorpe et al., 2012) and encodes for subunits of a voltage‐gated potassium channel. The tetrameric channel is responsible for M‐current which regulates neuronal excitability (Schroeder et al., 1998; Wang et al., 1998). Each subunit of the channel contains six transmembrane domains (S1–S6): the S1–S4 form a voltage‐sensing domain (VSD) while the S5–S6 participate in comprising a pore domain (Li et al., 2021). In addition, each subunit has a short intracellular N‐termini, and a long intracellular C‐termini which harbors three helices (HA, HB, and HC). HA and HB form a hairpin which could bind to a calcium sensor called calmodulin (CaM) (Ambrosino et al., 2015).

In this study, we reported the clinical features of 15 encephalopathy newborns each carrying a newly reported pathogenic variant, from a 113‐case cohort of unexplained NE. We depicted the therapeutic schedules and clinical outcomes of four patients affected by mutated KCNQ2 and also simulated the protein structure stability change induced by the KCNQ2 variants. We expect our study could provide valuable clues for the clinical molecular diagnosis and basic research in NE.

2. METHODS

2.1. Ethical compliance

This study was approved by the Ethics Committee of Hunan Children's Hospital (No. HCHLL‐2022‐43). Informed consent was obtained from the legal guardian of each newborn patient. All methods were performed in accordance with the Declaration of Helsinki.

2.2. Patients

113 neonates with encephalopathy of unknown cause were registered at the Division of Neonatology, Hunan Children's Hospital from January 2019 to May 2021. The initial symptoms, main clinical manifestations, other deformations, and the disease status and other conditions at each follow‐up were recorded. Patients received regular clinical tests including ammonia test, metabolic screening test, and amplitude‐integrated electroencephalographic (aEEG) monitoring at admission, and accepted brain Magnetic Resonance Imaging (MRI) scanning within 3–7 days after admission. Besides, all patients and their families underwent genetic test. Targeted panel sequencing (TPS) was chosen if an infant manifested a single and definite symptom of epilepsy, or had an abnormal testing result implying probable inborn error of metabolism (IEM). Otherwise, WES was adopted.

2.3. Amplitude‐integrated electroencephalographic monitoring

Electroencephalographic monitoring were measured using the aEEG classification system described by Lena Hellstrom‐Westas in 2006 (Hellstrom‐Westas & Rosen, 2006). According to this classification, the background pattern describing the dominating type of electrocortical activity in the aEEG trace could be divided into five categories including continuous normal voltage (CNV), discontinuous normal voltage (DNV), burst suppression (BS), continuous low voltage (CLV) and inactive, flat trace(FT). The CNV pattern indicated a normal aEEG result and DNV meant mild abnormality. Both BS and CLV manifested that the aEEG result was of moderate–severe abnormality. FT represented a severe abnormality. The results were reported by electrophysiological technicians independently.

2.4. Brain magnetic resonance imaging

The multimodal brain Magnetic Resonance Imaging (MRI) scanning was conducted by using Skyra 3.0T superconductivity MR scanner. The equipment was obtained from Siemens (Munich, Germany), with scanning sequence including axial T1 weighted image, axial T2 weighted image, axial T2 FLAIR image, DWI image, and the derived series apparent diffusion coefficient (ADC) image, using an eight‐channel phased‐array head coil. The slice thickness of each modality ranged from 3 to 5 mm.

2.5. DNA sequencing

EDTA anti‐coagulated venous blood was collected from neonates and DNA extraction experiment was implemented with the QIAamp DNA Blood Mini Kit. Whole exome enrichment was performed using xGen™ Exome Research Panel v2, and panel DNA fragment enrichment was conducted using AmpliSeq™ for Illumina Custom DNA Panel (Supplementary Materials File S1). Enriched DNA was sequenced with the Illumina NovaSeq® systems.

2.6. Bioinformatic analysis

Raw paired‐end reads were preprocessed via fastp (Chen et al., 2018) and clean reads were aligned to the human reference genome version 19 (hg19) using BWA MEN (Li & Durbin, 2009). Variants were called by HaplotypeCaller in GATK4.0 suite using standard hard filtering parameters according to the GATK Best Practices recommendations (Van der Auwera et al., 2013). Variant annotation was performed using ANNOVAR (Wang et al., 2010) and nonsynonymous variants were further evaluated using SIFT (Kumar et al., 2009), PolyPhen‐2 (Adzhubei et al., 2010), MutationTaster (Schwarz et al., 2010), REVEL (Ioannidis et al., 2016), and other pathogenicity prediction methods. Variant filtering was implemented with an in‐house bioinformatic pipeline.

2.7. Variant interpretation

The variant interpretation procedure was executed stringently under the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines (Richards et al., 2015), and its refinement and updated recommendations (Abou Tayoun et al., 2018; Biesecker & Harrison, 2018; Brnich et al., 2019; Ghosh et al., 2018). The presumptive causal variants were confirmed by Sanger sequencing, and searched against ClinVar (Landrum et al., 2018), OMIM (https://omim.org/), Human Gene Mutation Database(HGMD) (Stenson et al., 2009), and Google Scholar to confirm novelty.

2.8. Protein structure prediction

The protein structure file of KCNQ2 was obtained from the Protein Data Bank (Burley et al., 2021) with the accession number 7cr0. Change in stability of protein structure upon single point missense mutation was assessed using Dynamut2 (Rodrigues et al., 2021). Gibbs Free Energy change (ΔΔG) was introduced to measure the predicted stability change. A missense mutation was assumed to affect protein stability if ΔΔG < 0.0 kcal/mol. Residue interaction visualization results were generated online in Dynamut2. Protein structure prediction was performed online with RoseTTAFold (Baek et al., 2021).

3. RESULTS

3.1. Patients overview

The mean gestational age of the 113 enrolled neonates was 38.6 ± 1.5 weeks, and the mean birth weight was 2957.2 ± 560.7 g. In the cohort, 53.1% of the infants (60/113) had predicted pathogenic genetic defects including single nucleotide variation (48 cases), copy number variation (8 cases), and chromosome abnormality (4 cases). The 48 patients with identified pathogenic single nucleotide variation could be further classified into four subgroups: patient group affected by syndromic‐ (18/48, 37.5%), metabolic‐ (16/48, 33.3%), epileptic‐ (11/48, 22.9%) and mitochondrial‐related genes (3/48, 6.3%), according to the categories of encephalopathy gene proposed by Yang et al (Yang et al., 2020).

Among the NE cohort, 15 patients (P1‐P15, 8 males and 7 females) each carried an unreported pathogenic variant were selected for further analysis (Table 1). The parents of each newborn denied a family history of encephalopathy or other neurological diseases. Except for P2, P8, and P13, symptoms of all other patients started within the first 3 days after birth. The initial symptoms of the 15‐case sub‐cohort (Table 1 Column 10 underlined) were variable. Seven patients had seizure and another five patients showed decreased response to stimuli or were in coma at admission. Other clinical manifestations and additional congenital abnormalities were also observed (Table 1). Two patients were detected with excessive blood ammonia concentration (>1000 μmol/L) and four were diagnosed with abnormal metabolic screening results. The brain MRI results presented obvious symmetric patchy high‐intensity shadows in P7, P10, and P11 (Figure 1), and this kind of abnormality was reported to be related to inborn metabolic disorder (Biswas et al., 2021). Another seven patients presented minor subarachnoid hemorrhage, widening of cerebral space, or subarachnoid cyst which would not affect neurological development (Table 1, Supplementary Materials File S4). In the aEEG results, nearly a half of the patients (8/15) showed DNV pattern (mild abnormality) at onset. And the rest patients had moderate–severe abnormality (Table 1). From the last follow‐up records, six patients were dead. Specifically, P3, P4, and P9 died due to respiratory failure during 3–6 weeks after birth. Another three patients were in coma at admission and died for the same reason in the first 2 weeks after birth.

TABLE 1.

Information of 15 newborn patients with unexplained encephalopathy. Information including birth weight, onset age, sex, main clinical manifestation, other deformations, the brief results of regular clinical tests and genetic test, and other basic descriptions of 15 neonates with unexplained encephalopathy.

ID	Gestation (weekday)	Birth weight (g)	Onset age (days)	Length of stay (days)	Sex	Cesarean section	SGA	Pregnancy complications	Main clinical manifestation (initial symptoms were underlined)	Other deformations	Max blood ammonia (umol/L)	Metabolic screening	aEEG	Brain MRI	Outcome	Genetic test type	Variant information
P1	37	1950	1	37	M	Yes	Yes	Thalassemia	Dysphagia, jaundice	No	40	Normal	3	Mild enlargement of left ventricle	Neurodevelopmental delay	WES	CACNA1G (c.4023C > A)
P2	39+6	3300	16	55	M	Yes	No	No	Seizure, fever, respiratory distress	No	27.6	Normal	2	Widened frontotemporal extracerebral space	Normal	WES	NUP214 (c.3296G>A)
P3	40+6	2300	1	19	M	Yes	Yes	No	Decreased response to stimuli, tachypnea, poor sucking, hypotonia	Large tongue, hypospadias, cryptorchid, finger joint deformation	43.5	Normal	2	Widened temporal extracerebral space	Dead	WES	CHD4 (c.2239A>G)
P4	38	3850	1	23	M	Yes	No	No	Poor sucking, respiratory distress	Full moon face, small jaw, foot deformity	35	Normal	2	Normal	Dead	WES	HNRNPK (c.886C>T)
P5	38+3	3180	1	31	F	Yes	No	No	Seizure	No	36.5	Normal	2	Bilateral subependymal cysts	Normal	TPS (Epilepsy)	CC2D2A (c.3497A>T)
P6	37+6	2650	1	13	F	Yes	No	No	Poor sucking, fever, respiratory distress	Ocular hypertelorism, small palpebral fissure	64.9	Normal	2	Normal	Neurodevelopmental delay	WES	ERF (c.90G>A)
P7	39+5	3250	1	11	M	No	No	No	Coma, respiratory distress, hypotonia	No	39.4	Slightly elevated Glycine	4	Multiple abnormal signals in the bilateral basal ganglia, hypoplastic corpus callosum, internal capsule and brainstem	Dead	WES	MRPL3 (c.413_414del)
P8	37+5	3000	21	37	F	No	No	No	Seizure	No	21.7	Ketonuria	3	Widened temporal extracerebral space	Normal	TPS (Epilepsy)	COQ2 (c.411G>A)
P9	40	1950	1	38	M	No	Yes	No	Decreased response to stimuli, thrombocytopenia, refractory acidosis, abdominal distension	No	38	Normal	3	Normal	Dead	WES	BCS1L (c.321‐2A>G)
P10	39+3	2800	1	5	F	No	No	No	Coma, dystonia	No	1482.6	OTCD/CPS1D	4	Symmetrical short T1, long T2 signal and diffusion limited in bilateral basal ganglia and thalamus	Dead	WES	CPS1 (c.1781G>T)
P11	38+3	3150	2	5	F	No	No	No	Coma, jaundice, hypotonia, groan	No	1103	MMA/PA	4	Symmetrical patchy high‐intensity shadows in bilateral basal ganglia, thalamus, corpus callosum, brainstem and cerebellar dentate nucleus	Dead	TPS (IEM)	MMUT (c.929delG)
P12	39	3600	3	37	F	Yes	No	No	Seizure	No	65	Normal	2	Normal	Normal	TPS (Epilepsy)	KCNQ2 (c.436T>C)
P13	37	3200	5	18	M	Yes	No	No	Seizure, mild acidosis	No	26.6	Normal	3	Small amount of subarachnoid hemorrhage	Neurodevelopmental delay	WES	KCNQ2 (c.545T>C)
P14	40+5	3550	3	24	F	No	No	No	Seizure, decreased response to stimuli, respiratory distress	No	89.9	Normal	2	Small amount of subarachnoid hemorrhage	Normal	WES	KCNQ2 (c.911T>A)
P15	37+5	3800	2	38	M	No	No	Hypertension	Seizure	No	75.9	Normal	2	Normal	Neurodevelopmental delay	TPS (Epilepsy)	KCNQ2 (c.1220_1223del)

Note: The Length of stay includes the days within the authors' hospital, and the days in other institution(s) before transferred to the authors' hospital.

In the column of aEEG, numbers 2–4 represent discontinuous normal voltage(DNV), burst suppression(BS) and continuous low voltage(CLV), respectively.

The brain MRI results of P7, P10 and P11 are abnormal (Figure 1) and P4, P6, P9, P12 and P15 are normal. The brain MRI results of the rest seven patients are curated in Supplementary Materials File S4.

The details of Panel (Epilepsy) and Panel (IEM) are described in Supplementary Materials File S1.

Abbreviations: aEEG, amplitude‐integrated EEG; CPS1D, carbamoyl phosphate synthetase deficiency; IEM, inborn errors of metabolism; MMA, methylmalonic acidemia; MRI, magnetic resonance imaging; OTCD, ornithine transcarbamylase deficiency; PA, propionic acidemia; SGA, small for gestational age; TPS, targeted panel sequencing; WES, whole exome sequencing.

FIGURE 1.

Brain MRI results of P7, P10, P11 and P15. The brain MRI results of P7, P10, P11 and P15 with each line representing an image series of one patient. P7, P10 and P11 is abnormal and P15 is normal in MRI results.

3.2. Variants overview

Each of the 15 patients was diagnosed with a single newly discovered pathogenic genetic alteration (Table 2). These variants (V1–V15) were distributed in nine different chromosomes (Figure 2), and belonged to 12 genes which could be classified into four groups (Table 2) according to the categories of encephalopathy gene proposed by Yang et al. (2020). In general, nine variants were rated as “Pathogenic” (V4, V11, and V15) or “Likely Pathogenic” (V3, V6, V7, V8, V9, and V12), with the others rated as VUS (Variants of Uncertain Significance). As with the source of variant, eight were de novo (Table 2). Moreover, three variants were included in the gnomAD (Lek et al., 2016) with frequencies lower than 1‰, with all other variants absent. All variants were confirmed by Sanger sequencing (Supplementary Materials File S2).

TABLE 2.

Information of 15 newly discovered variants related to NE. Information including encephalopathy gene category, ACMG/AMP classification and evidence, variant source, population variant frequency, pathogenicity prediction results and other basic descriptions of 15 newly discovered variants related to NE.

ID	Gene	Category	Transcript & Exon	Variant	ACMG/AMP Classification & Evidence	Source	gnomAD V2.1.1	Pathogenicity prediction
								SIFT	PolyPhen‐2	MutTaster	REVEL
V1	CACNA1G	Syndromic	NM_018896.5 exon21	c.4023C>A (p.Y1341*)	VUS PS2_Moderate, PM2	De novo	—	—	—	A	—
V2	NUP214	Syndromic	NM_005085.4 exon23	c.3296G>A (p.R1099Q)	VUS PM2	Maternal	0.0001	T	D	D	0.253
V3	CHD4	Syndromic	NM_001273.5 exon15	c.2239A>G (p.T747A)	Likely Pathogenic PS2, PM2, PP3	De novo	—	T	D	D	0.758
V4	HNRNPK	Syndromic	NM_031263.4 exon11	c.886C>T (p.R296*)	Pathogenic PVS1, PS2, PM2	De novo	—	—	—	A	—
V5	CC2D2A	Syndromic	NM_001378615.1Exon28	c.3497A>T (p.D1166V)	VUS PM2	Paternal	2.55 × 10−5	D	D	D	0.726
V6	ERF	Syndromic	NM_006494.4 exon2	c.90G>A (p.W30*)	Likely Pathogenic PVS1, PM2	Maternal	—	—	—	A	—
V7	MRPL3	Mitochondrial	NM_007208.4 exon4	c.413_414del (p.C138*)	Likely Pathogenic PVS1, PM2	Maternal	3.98 × 10−6	—	—	—	—
V8	COQ2	Metabolic	NM_001358921.2exon2	c.261G>A (p.W87*)	Likely Pathogenic PVS1, PM2	Paternal	—	—	—	A	—
V9	BCS1L	Metabolic	NM_001079866.2 Intron2	c.321‐2A>G	Likely Pathogenic PVS1, PM2	Paternal	—	—	—	D	—
V10	CPS1	Metabolic	NM_001875.5 exon16	c.1781G>T (p.G594V)	VUS PS2_Moderate, PM2, PP3	De novo	—	D	D	D	0.965
V11	MMUT	Metabolic	NM_000255.4 exon5	c.929del (p.G310Efs*4)	Pathogenic PVS1, PM2, PM3	Maternal	—	—	—	—	—
V12	KCNQ2	Epileptic	NM_172107.4 exon3	c.436T>C (p.W146R)	Likely Pathogenic PS2, PM2, PP3	De novo	—	D	D	D	0.898
V13	KCNQ2	Epileptic	NM_172107.4 exon4	c.545T>C (p.V182A)	VUS PS2_Moderate, PM2, PP3	De novo	—	D	B	D	0.88
V14	KCNQ2	Epileptic	NM_172107.4 exon6	c.911T>A (p.F304Y)	VUS PS2_Moderate, PM2, PP2, PP3	De novo	—	D	D	D	0.738
V15	KCNQ2	Epileptic	NM_172107.4 exon11	c.1220_1223del (p.K407Tfs*32)	Pathogenic PVS1, PS2_Moderate, PM2	De novo	—	—	—	—	—

Note: For SIFT: D: Deleterious; T: Tolerated; For PolyPhen‐2: D: Probably Damaging; P: Possibly Damaging; B: Benign. For MutationTaster: A: Disease_causing_automatic; D: Disease_causing; N: Polymorphism; P: Polymorphism_automatic.

FIGURE 2.

Variant distribution map. Illustration of chromosome distribution of 15 newly discovered variants (V1–V15) related to NE. The symbol of the gene which a variant belongs to is in brackets.

3.3. Patients with mutated KCNQ2

Four patients (P12–P15) were affected by KCNQ2 variants in the sub‐cohort (Table 2). P12 carried a missense mutation (V12: W146R). Convulsion started at the 4th day after birth and then symptoms had been controlled for about a month by oral phenobarbital (PB) (Figure 3). The patient had seizure recurrence for two times with each lasting for 5–10 s and was then relieved without any intervention during the next 3 months. She had been seizure‐free from the 4th month to the last follow‐up in the 12th month.

FIGURE 3.

Therapeutic schedules of the four patients (P12‐P15) affected by mutated KCNQ2. PB, phenobarbital; OXC, oxcarbazepine; LEV, Levetiracetam; VPA. Valproate.

P13 hold a V182A mutation (V13) and started to convulse at the 5th day after birth. This patient had a moderate–severe abnormality in brain waves and received PB for nearly 1 month (Figure 3). After symptom‐controlled, the patient discharged from hospital. He received no subsequent treatment until seizure relapse. Oxcarbazepine (OXC) was administered orally to the patient since the 6th month and stopped without order in the end of the 7th month. Neurodevelopmental delay eventually occurred in this patient.

P14 carried a F304Y variant (V14) and developed seizure at the 3rd day after birth. Decreased response to stimuli and respiratory distress were also observed in this patient. The infant took PB for 2 months (Figure 3). Besides, Levetiracetam (LEV) was added to control symptoms from the 13th day to the 20th day. Normal growth and neurological development were informed in the regular follow‐ups until the 12th month.

P15 holds a null variant (V15: K407Tfs*32), and his mother had pregnancy‐induced hypertension. This infant developed seizure at the second day after birth but the brain MRI result was normal (Figure 1) which was consistent with the case descriptions in other reports (Laccetta et al., 2019; Legros et al., 2022). P15 was treated with PB during neonatal period and sequenced with valproate (VPA) due to its better efficiency in controlling symptoms. The neurodevelopmental delay was finally occurred even though the patient was persistently seizure‐free with oral VPA.

3.4. The details of KCNQ2 variants

V12 (W146R) was located in the linker of segment 2 (S2) and segment 3 (S3) transmembrane domain of the KCNQ2 subunit (Figure 4a). This mutation resulted in a Tryptophan‐to‐Arginine substitution at codon site 146, and the disappearances of hydrophobic contacts between the indolyl group of Tryptophan and other residues nearby (Figure 4b, green dashed line), as well as a Van der Waals interaction between W146 and R144 (Figure 4b, light blue dashed line). The prediction result of protein stability change (ΔΔG = −1.34 kcal/mol) suggested that V12 might destabilize the protein structure.

FIGURE 4.

The illustrated locations of the four KCNQ2 variants, and the predicted residue interaction states and the conservativeness of the 146th, 182th and 304th position of the KCNQ2 protein. (a) Schematic representation of KCNQ2 subunit and the illustrated positions of the four KCNQ2 variants (V12–V15). The subunit contains six transmembrane domains (S1–S6): the S1–S4 form a voltage‐sensing domain (VSD) while the S5–S6 participate in comprising a pore domain. In addition, the subunit has a short intracellular N‐termini and a long intracellular C‐termini which harbors three helices (HA, HB and HC). The positions of the four KCNQ2 variants are pointed out with red arrows. The position of the premature termination introduced by V15 (K407Tfs*32) is denoted as red vertical line. (b–d) show the predicted residue interaction states of the 146th, 182th and 304th position of the wild‐type and the mutated KCNQ2 protein, respectively. Hydrogen bond, Van der Waals interaction and hydrophobic contact are denoted as red, light blue and green dashed line, respectively. (e–f) show the sequence concordance analysis results of the 146th, 182th and 304th position of the KCNQ2 protein, respectively.

V13 (V182A) and V14 (F304Y) resided in the S3 and S6 helical segment, respectively. S3 participates in forming the voltage‐sensing domain (VSD) while S6 is a part of the central pore domain (Li et al., 2021). V13 led to the cancels of two hydrophobic contacts between residue 182 and 87 (Figure 4c, green dashed line), and one hydrogen bond between residue 182 and 178 (Figure 4c, red dashed line). As with V14, Tyrosine replaced Phenylalanine in the 304th position of the amino acid sequence of KCNQ2. This event altered the hydrophobic contact pattern within the space around residue 304 (Figure 4d, green dashed line). Furthermore, two new hydrogen bonds were formed in comparison to the wild‐type structure (Figure 4d, red dashed line). The ΔΔGs were −0.5 and −0.13 kcal/mol for V13 and V14, respectively.

All the three point mutations were predicted to bring about instability to the KCNQ2 protein structure. Besides, the results of sequence concordance analysis (Figure 4e–g) revealed that the 146th, 182th and 304th position of KCNQ2 protein were highly conserved between human (Homo sapiens) and other species including house mouse (Mus musculus), Norway rats (Rattus norvegicus), Rhesus macaques (Macaca mulatta), Dog (Canis lupus familiaris) and Zebra finch (Taeniopygia guttata). This strong conservativeness indirectly implied that the nucleotide alterations of the three sites might go against the normal functioning of the KCNQ2 protein.

V15 (K407Tfs*32) was a frameshift mutation situated between HA and HB of the long C‐terminal tail (Figure 4a). At codon site 438, this variant was predicted to introduce a premature termination defect (Figure 4a, red vertical line) which led to a truncated or absent protein. Structural simulation exhibited an significant discrepancy between wild‐type protein and the truncated KCNQ2 (Supplementary Materials File S3).

4. DISCUSSION

This study focused on 15 NE patients each with a newly reported pathogenic variant. These variants belonged to 12 genes among which KCNQ2 was reported as the most frequently affected gene related to NE (Yang et al., 2020). The therapeutic schedules and clinical outcomes of the four patients affected by the pathogenic KCNQ2 variants were depicted in detail and the resultant protein structure stability changes were predicted.

However, there were two major limitations in our work and we elucidate them as below:

1. Up to six variants were tagged with VUS in this study. Generally, a variant is rated as VUS for two reasons: (i) currently, no report or information from related database records the clinical significance of the variant; (ii) there is a disagreement in the clinical significance of the variant. Hence, with science advance and data accumulation in the field of genetics and genomics, some VUS have the chance to be verified as the truly disease‐causing variants in the future. At the time of this writing, the six VUS were able to account for the corresponding clinical findings in our study according to the comprehensive judgment based on the combination of patient phenotype, clinical test results, family history, and previous knowledge. In the future, we will continuously follow these VUS.

2. Predicted to introduce a premature termination defect at codon site 438, V15 (K407Tfs*32) is a frameshift mutation located in the C‐terminal tail of KCNQ as mentioned in the Result section. Since experimentally determined complete KCNQ2 structure is not available at present, we employed RoseTTAFold (Baek et al., 2021) to predict the full‐length structures of the wild‐type KCNQ2 and the truncated KCNQ2 caused by V15 in a ab‐initio way. Although the structural simulation results exhibited an significant discrepancy between the two (Supplementary Materials File S3), it might be far from reflecting the real situation after manual inspections. Breakthroughs have been made in the past decade in the field of computational prediction of protein structure (Braberg et al., 2022), but many challenges remain especially the those about predictive accuracy (David et al., 2022).

Comparing with the wild‐type KCNQ2 protein, the truncated mutant in our study lost HB and HC in the C‐terminal tail. HB participates in binding to a calcium sensor called calmodulin (CaM) with HA (Ambrosino et al., 2015). Binding of CaM is considered essential in folding and trafficking of KCNQ2 (Strulovich et al., 2016) and defect in HA and HB has been reported to impair the surface expression (Zhou et al., 2016). HC has been reported to mediate subunit assembly (Baculis et al., 2020; Haitin & Attali, 2008). Hence, the channel maturation and plasma membrane expression of KCNQ2 were inferred to be hampered in P15. Irreversible neurodevelopmental delay was developed in this patient in spite of medically controlled seizure. P13 also suffered from mental development delay; however, the situation of this patient was quite different. The pathogenic missense variant of P13 resided in the S3 helical segment (Figure 4) which participates in forming the voltage‐sensing domain (VSD) of KCNQ2. The patients did not take anticonvulsants drugs on time as the doctors recommended and the parents refused follow‐up. This negative attitude to treatment and communication may be the principal reason for the final poor neurological outcomes. Aside from P13 and P15 both with negative outcomes, P12 and P14 were normal in growth and neurological development as recorded in the last follow‐ups. The pathogenic point mutations of P12 and P14 were located in the intracellular linker of S2 and S3, and S6 helical segment, respectively (Figure 4). Goto et al. conducted a comprehensive analysis on the characteristics of KCNQ2 variants causing either benign familial neonatal epilepsy (BFNE) or developmental and epileptic encephalopathy (DEE) with in‐house data and a large amount of public data (Goto et al., 2019). They revealed that in the comparison between BFNE and DEE sets, BFNE missense variants frequently localized to the intracellular domain between S2 and S3. This regional specificities is in consistence with our finding in the situation of V12. Besides, S6 was inferred as a hotspot region for DEE mutations by two independent research teams (Goto et al., 2019; Zhang et al., 2020). However, in our case, the epilepsy of P14 who carried a pathogenic variant in this domain is benign. In general, the limit amount of cases with pathogenic KCNQ2 variant in our study obstructed the pattern validation and discovery in variant location's impact on the patient phenotype or disease outcome.

NE brings heavy financial burden and tremendous mental pressure to the patients’ family and early diagnosis is essential for optimizing the therapeutic schedule and strategy for patients. However, related genetic factors underlying this newborn illness are complicated, making accurate diagnosis challenging. Data accumulation in both clinical and genetics will be conducive to elucidate the disease pathogenesis, providing convenience for diagnosis and treatment in the future. In this study, 15 NE‐related pathogenic variants are reported for the first time, which expand the mutation spectrum of genes involved in pediatric nervous system diseases, and introduce new evidence for molecular diagnosis in encephalopathy of neonates.

AUTHOR CONTRIBUTIONS

Rong Zhang, Jingjing Xie and Xiaoming Peng designed the study. Rong Zhang, Jingjing Xie, Yan Zhuang, Fan Zhang, Weiqing Huang, Min Zhang and Junshuai Li performed data collection. Xiao Yuan, Jianfei Hou and Rong Zhang performed data analysis. Xiao Yuan, Yanqin Liu and Jianfei Hou performed plotting. Rong Zhang and Xiao Yuan prepared the manuscript. Yan Yu, Qiang Gong and Xiaoming Peng revised the manuscript. All authors reviewed and approved the manuscript.

CONFLICT OF INTEREST STATEMENT

X.Y., Y.Y., J.H., Y.L., and Q.G. are all employees of Changsha Kingmed Center for Clinical Laboratory. All other authors declare no conflict of interest.

Supporting information

File S1.

File S2.

File S3.

File S4.

Zhang, R. , Xie, J. , Yuan, X. , Yu, Y. , Zhuang, Y. , Zhang, F. , Hou, J. , Liu, Y. , Huang, W. , Zhang, M. , Li, J. , Gong, Q. , & Peng, X. (2024). Newly discovered variants in unexplained neonatal encephalopathy. Molecular Genetics & Genomic Medicine, 12, e2354. 10.1002/mgg3.2354

DATA AVAILABILITY STATEMENT

The information of the 15 novel variants (V1–V15) discovered in this study had been submitted to ClinVar and the corresponding accession numbers were SCV003842160‐SCV003842174. Other datasets generated during and/or analyzed during the current study are not publicly available because of ethical and privacy restrictions, but are available from the corresponding author on reasonable request.