Mutations obstructing ATP's emplacement in KIF2A nucleotide‐binding pocket causes parenchymal malformations, motor developmental delay, with intellectual disability

Abstract

Background

KIF2A‐related tubulinopathy (MIM: #615411) is a very rare disorder that was clinically characterized as microcephaly, epilepsy, motor developmental disorder (MDD), and various malformations of cortical development, but intellectual disability (ID) or global developmental delay (GDD) was rarely reported in the patients.

Methods

Quad whole‐exome sequencing (WES) was performed on the proband, the older brother, and their parents. Sanger sequencing was used to verify the candidate gene variant.

Results

The proband, a 23‐month‐old boy, was previously diagnosed with GDD, and his brother, aged nine years, had ID; both were born to a healthy couple. Quad‐WES detected a novel heterozygous KIF2A variant, c.1318G>A (p.G440R), in both the brothers but not in the parents. In‐silico analysis revealed that the variants G440R and G318R (which were previously reported in the only reported patient with GDD) lead to markedly enlarged side chains and hinder ATP's emplacement in the NBD pocket.

Conclusions

The type of KIF2A variants that sterically hinder ATP emplacing in KIF2A NBD pocket may be associated with the intellectual disability phenotype; however, further studies are needed. Findings in this case also suggest a rare parental germline mosaicism of KIF2A G440R.

Compared to the known KIF2A mutations that putatively hinder ATP docking by changing the hydrophily or amphiphile of the residues, a substitution of glycine to arginine spatially blocks the ATP's emplacement in the KIF2A nucleotide‐binding pocket.

1. BACKGROUND

Complex cortical dysplasia with other brain malformations 3 (CDCBM3, MIM: #615411) is a very rare type of tubulinopathies caused by a heterozygous mutation in the kinesin family member 2a (KIF2A) encoding gene. No more than 10, as far as we know, cases of CDCBM3 were previously reported globally (Hatano et al., 2021). Hatano et al. (2021) concluded that patients with CDCBM3 usually had agyria/pachygyria (lissencephaly) with severe motor dysfunction, microcephaly, and early‐onset epilepsy; Costain et al. (2019) and Yuen et al. (2015), in separate studies, identified heterozygous KIF2A variants in patients diagnosed with epilepsy or autism spectrum disorder (ASD), respectively; however, no mental developmental delay (MDD) or intellectual disability (ID) was documented in these cases.

KIF2A belongs to the human kinesin‐13 family (M‐kinesins) consisting of KIF2A, KIF2B, KIF2C/MCAK, and KIF24 (Miki et al., 2005), and it functions in neuronal migration, axonal elongation, and pruning (Broix et al., 2018; Homma et al., 2018; Maor‐Nof et al., 2013). Kif2a‐null mice showed multiple neurodevelopmental abnormalities like early postnatal death, cortical lamination defects as a result of aberrant neuronal migration, and lateral cortical ventricle enlargement (Homma et al., 2003). Interestingly, all reported causative KIF2A variants, except for R723H found in a study of ASD (Yuen et al., 2015), in humans are located in the kinesin motor domain of the KIF2A protein (amino acid residues 223–553), indicating a critical role of this domain in CDCBM3.

Herein, we report a novel KIF2A variant, G440R, in two brothers, which was tested with an inheritance of ‘de novo’. Both of the brothers had MDD or ID, departed to the CDCBM3 phenotype, that had never been reported in previous cases of CDCBM3.

2. METHODS

A 23‐month‐old boy was admitted to our hospital because of an out‐patient diagnosis of global developmental delay (GDD), including cognitive and motor developmental delay with delayed language ability. He was the second child born to healthy parents, and his older brother, aged 9 years, had an intellectual disability (ID) and was educated with a special education program.

Written informed consent was obtained from the guardians, and the study was approved by the Ethics Committee of Hainan General Hospital (approval number Med‐Eth‐Re [2022] 738).

Whole‐exome sequencing (WES) was performed on the patient, his brother, and their parents by the Chigene Translational Medical Research Center Co. Ltd. (Beijing, China). A whole exome library was constructed using the xGen Exome Research Panel v1.0 (IDT, Iowa, USA) and high‐throughput sequencing was performed on the NovaSeq 6000 sequencer (Illumina, San Diago, USA) with a short‐read method (PE150). The paired‐end reads were performed using Burrows‐Wheeler Aligner (BWA) and aligned to the Ensemble GRCh37/hg19 reference genome. Base quality score recalibration, together with SNP and short indel calling, was conducted using GATK. Yielded variants and their pathogenicity were automatically annotated using an independent‐developed online system (http://chigene.mysxl.cn/#advantages) provided by the Chigene company. The pathogenicity of genetic variants was classified into five classes, pathogenic, like pathogenic, uncertain significance, like benign, and benign, according to the American College of Medical Genetics (ACMG) clinical practice guidelines. The candidate variants were confirmed by Sanger sequencing in the family.

Evolutionary conservation analysis of the KIF2A residue was performed, and the results were curated by the UCSC genome browser (https://genome.ucsc.edu). The analysis of a three‐dimensional (3D) model of the KIF2A protein chain (PDB id: 5mio) was performed using PyMol, version 2.5.0, compiled for MAC OS.

Keyword “KIF2A” was used to search reported cases due to germline mutation in databases OMIM, PubMed, and Google Scholar.

3. RESULTS

3.1. Clinical findings

3.1.1. The proband

The patient had no significant facial or body deformation, and his vital signs (body temperature, pulse rate, and respiration) were normal. He was born at 40 weeks of gestation after an uneventful pregnancy. At his birth, the patient was 2.8 kg (−1.75 SD) in weight, 44.1 cm (<−3 SD) in body length, and 31 cm (2.54 SD) in head circumference. In the first six months after his birth, no disorder of movement, breath, or feeding difficulty was found. At the age of 14 months, the patient was found to be unable to sit independently. At age 23 months, he was taken to see a doctor, and the physical examination showed he had a body weight of 9.4 kg (−2.13 SD) and a head circumference of 44.5 cm (<−3 SD) with a closed bregma (anterior fontanelle). The patient kept drooling, and he could not speak but responded to his name by gazing and smiling. He could only walk a short distance with an unsteady and broad gait without assistance. The patient could understand and follow several simple commands.

Brain magnetic resonance imaging (MRI) showed his bilateral lateral ventricles were slightly enlarged, more significant on the left side; his cerebral gyrus was slightly widened (Figure 1). The Gesell assessment suggested the patient had mild to moderate global developmental delay (GDD) with profound language developmental delay. The patient had a language developmental age (DA) of 10.5 months and a developmental quotient (DQ) of 45; his adaptability DA was of 13.3 months and his DQ was of 57. He had a gross motor DA 15.63 months and a DQ of 67, fine motor DA of 14.47 months and DQ of 62; and a personal‐social DA of 17.5 months and a DQ of 75. In summary, the patient had a developmental quotient (DQ) equivalent to 13 months of development.

FIGURE 1.

Brain magnetic resonance imaging (MRI) findings in the proband and his brother.

After being treated with physical and language rehabilitation and supportive therapy for 5 months, the patient's ability to walk improved. At the age of 2 years and 4 months, he could walk without assistance for a longer distance, and he could say “baba” and “mama,” use nodding or shaking head in agreement or disagreement, respectively, and indicate requirements using finger pointing.

3.1.2. The brother of the proband

The patient's older brother, aged 9 years, also had GDD after his birth. An intelligence quotient (IQ) test, based on the Wechsler Intelligence Scale for Children (WISC), showed his IQ score was 26. The brother walked unstably, and he had severe cognitive impairment and speech impairment at the age of 20 months. At age 9 years, the brother was able to accomplish daily routines like eating, dressing, and going to the toilet by himself and he could follow simple instructions, but he could not speak except to say “baba” and “mama” occasionally.

When the brother was 10 months old, a brain MRI showed he had slightly wider gyrus, thin fibers, and a thin corpus callosum (Figure 1). An electroencephalogram showed epileptiform discharges; however, he had no history of seizures or use of anti‐epilepsy drugs.

Quad‐WES found a novel heterozygous variant, NM_001243952.2: c.1318G>A/p.G440R, in the KIF2A gene in both the brothers (Figure 2a), which is pathogenic, and has an evidence class of PS2 + PM1 + PM2 + PP1 + PP3, according to the ACMG guidelines. None of the other causative variants were found in known DD‐associated genes. Conservative analysis showed KIF2A residue G440 is highly evolutionarily conserved in multiple species (Figure 2b) and is a catalytic residue (Figure 2c). In comparison to all previously reported KIF2A mutations (Table 1), G440R and G318R (reported in a Chinese case; Cheng et al., 2022) resulted in remarkably enlarged side chains of amino acid residues (Figure 3a); 3D‐structure analysis indicated that the G440R variant obstructs the emplacement of the adenosine triphosphate (ATP) analog, AMP‐PNP, in the pocket of the nucleotide‐binding domain (NBD); for G318R, we had the same result. See Figure 3b.

FIGURE 2.

Confirmation and analysis of NM_001243952.2: c.1318G>A/p.G440R found in this study. (a) Sanger sequencing confirms de novo c.1318G>A in the family. (b) KIF2A residue G440 is highly conserved in multiple species. (c) KIF2A residue G440 is a catalytic residue.

TABLE 1.

Clinical features and KIF2A variants in 11 patients previously reported and in present studies.

Patient	Diagnosis	Sex	Age at diagnosis	Motor disabilities	DD/ID	NS	Anomaly	Cortical malformations	KIF2A variant	Reference
1	CDCBM3	M	23 m	Profound, difficulty walking with unsteady and broad gait	GDD with speech and cognition impairment and poor communication kills	Drooling	Mild growth retardation, microcephaly	Slightly enlarged bilateral lateral ventricles and a slightly widened cerebral gyrus	c.1318G>A (p.G440R)	Present study
2	CDCBM3	M	9 y	Profound, difficulty walking	ID with severe cognition and speech impairment	EEG showed epileptiform discharges.	–	Slightly wider gyrus, thin fibers, and thin corpus callosum	c.1318G>A (p.G440R)	Present study
3	CDCBM3	F	4.8 y	Profound	–	Seizures, spastic tetraplegia, and cortical blindness	Growth retardation, microcephaly, small ears, a mildly enlarged nose bridge, thin and tented lips, and scoliosis	Posterior agyria, thin corpus callosum	c.961C>G (p.H321D)	Cavallin et al. (2017)
4	CDCBM3	F	8 y	Profound	–	Seizures, spastic tetraplegia, and cortical blindness	Mild growth retardation, microcephaly, a slightly long nose, and a mildly short philtrum	Posterior agyria, frontal pachygyria	c.950G>A (p.S317N)	Cavallin et al. (2017)
5	CDCBM3	F	NM	Profound	–	Seizures, downward gaze, nystagmus, and ptosis	Unilateral microphthalmia and PHPV	Posterior agyria, frontal pachygyria, dysplastic corpus callosum	c.959C>T (p.T320I)	Tian et al. (2016)
6	CDCBM3	F	0.8 y	Profound	–	Absent seizures, spastic tetraplegia, and axial hypotonia	Microcephaly, round face, subtle synophrys, and thin and tented lips	Posterior agyria, frontal pachygyria	c.950G>A (p.S317N)	Poirier et al. (2013)
7	CDCBM3	M	13 y	Profound	–	Seizures, spastic tetraplegia, axial hypotonia, and nystagmus	Mild growth retardation and scoliosis	Posterior agyria, frontal pachygyria	c.962A>C (p.H321P)	Poirier et al. (2013)
8	Epilepsy	F	NM	NM	–	Seizures	NM	Delayed myelination, cerebral atrophy, thin corpus callosum	c.1363C>G (p.L455V)	Poirier et al. (2013)
9	ASD	NM	NM	NM	NM	NM	NM	NM	c.2168G>A (p.R723H)	Yuen et al. (2015)
10	CDCBM3	M	7 y	Profound, standing without assistance at age 8	–	Seizures, hypotonia	Growth retardation, microcephaly, and scoliosis	Posterior dominant pachygyria	c.1298C>A (p.S433Y)	Hatano et al. (2021)
11	CDCBM3	M	14 m	Profound	Severe mental DD with speech impairment and poor communication skills		Congenital laryngomalacia	Anencephaly	c.952G>A (p.G318R)	Cheng et al. (2022)

Abbreviations: ASD, autism spectrum disorder; DD, developmental delay; ID, intellectual disability; m, months; MDD, motor developmental delay; NM, not mentioned; NS, nervous system; PHPV, persistent hyperplastic primary vitreous; y, year(s).

FIGURE 3.

Analysis of KIF2A variants G440R and G318R compared to the other previously reported causative variants. (a) In eight types of substitution of amino acid residues in the variants, the conversion of glycine to arginine results in a markedly enlarged amino acid side chain, which (b) hinders the emplacement of ATP (as shown with the ATP analog, AMP‐PNP) in both G440R and G318R. The purple spherical molecules show the localization of ATP molecules on the surface of the KIF2A protein. The red blocks show that all predicted arginine side chains are sterically hindered by the ATP molecule.

Because the parents did not carry the variant, a putative theory of germline mosaicism or gonadal mosaicism of KIF2A c.1318G>A explains the results.

Seven variants in the KIF2A gene were found in eight patients in the previously reported cases, in which KIF2A S317N was identified in two unrelated individuals. All the reported causative variants were missense mutations and, however, the clinical data in Case 8 found R723H were unavailable (Costain et al., 2019). Clinical features and genetic findings in 11 patients, including ours, are listed in Table 1.

4. DISCUSSION

Complex cortical dysplasia with other brain malformations 3 (CDCBM3, MIM: #615411) is an autosomal dominant tubulinopathy characterized as microcephaly, nystagmus, seizures, motor developmental delay, cerebral gyrus hypertrophy, intrauterine fetal growth retardation, and multiple parenchymal malformations, including corpus callosum hypoplasia, spastic tetraplegia, cortical dysplasia, anencephaly, and subcortical banding heterotopia. However, there have been few previous reports on the evaluation of cognitive impairment or intellectual disability in patients with CDCBM3, except for a recently reported Chinese case (Cheng et al., 2022). In three Chinese patients, including two in our study, severe cognitive impairments and speech disabilities were found apart from profound motor developmental delay (Cheng et al., 2022), indicating the same causative factor.

Motor and intellectual disabilities are common in patients with tubulinopathies, which appear to correlate positively with the degree of brain malformation (Bahi‐Buisson & Maillard, 1993), while epilepsy was not necessarily determined by the severity of the cortical malformation in most of the patients (Romaniello et al., 2019). In previously reported CDCBM3 cases, six out of nine patients had posterior agyria or pachygyria, of which four had frontal pachygyria, three had thin/dysplastic corpus callosum, and one had delayed myelination cerebral atrophy (Table 1). In comparison to the reported Chinese patient (Cheng et al., 2022) (and one was diagnosed with autism spectrum disorder without clinical data; Yuen et al., 2015), who had anencephaly, the other seven patients all had epileptic seizures (Table 1), suggesting different physiological bases. In three, including ours, Chinese patients with CDCBM3, significant variability in the character and extent of the brain parenchymal malformations could not explain the shared cognitive impairment or intellectual disability or the lack of epileptic seizures.

Hatano et al. (2021) concluded that previously reported KIF2A variants, S317N, T320I, H321D, and S433Y, are all located on the nucleic‐binding domain (NBD) or ATP‐binding region (residues 313–320) and/or at the pocket for ATP docking, which might be causative by changing the hydrophily or amphiphily of the residues (Hatano et al., 2021). We found the two variants, G318R and G440R, do not occur in the ATP‐binding region but are spatially adjacent to the ATP molecular, and further, the substitution of glycine to arginine results in a markedly enlarged residue side chain and is sterically difficult to avoid blocking ATP's emplacement compared to the other missense variants (Figure 3a,b). Thus, our findings support that the interruption of ATP microtubule binding would lead to a non‐functional KIF2A (Poirier et al., 2013). KIF2A is an ATP‐driven microtubule‐associated protein that uses the energy of ATP hydrolysis to carry out mechanical work along microtubule tracks (Ali & Yang, 2020; Poirier et al., 2013), and, in zebrafish larvae, kif2a knockout resulted in reduced head size and neuronal loss in the brain due to a decrease in cell proliferation and increased levels of cell apoptosis (Partoens et al., 2021), which supported the ATP‐driven function theory.

However, we did not know whether more severe brain malformations or epilepsy were associated with a complete loss of ATP‐binding function. In this study, it was found that G440R was located at the bottom of the ATP spatial position (Figure 3b), which may have affected the local conformation of the ATP‐NBD complex, but it may not be enough to cause a complete loss of the energy release function of ATP hydrolysis; in comparison, G318R found in another Chinese case (Cheng et al., 2022) is located on the NBD, that is, the ATP‐binding region, so it also affects the chemical bonding of ATP and NBD while hindering the emplacement, which may explain the severer parenchymal malformations.

Finding of the same “de novo” variant in both the siblings, suggesting a parent germline mosaicism in this case. Germline mosaicism is a relatively frequent mechanism of inherited disease and provides an explanation for the inheritance pattern in cases where multiple affected offspring are born to healthy parents, and recent next‐generation sequencing methods have improved the sensitivity to detect mosaic events (Avigdor et al., 2021). However, we could not find out if KIF2A variant G440R was paternal or maternal, and, thus, an early prenatal diagnosis would be necessary for further pregnancy in this family.

5. CONCLUSIONS

Findings in this study revealed that mental developmental delay, or ID is a critical phenotype in CDCBM3 that may be caused by sterically hindering ATP's emplacement into KIF2A.

AUTHOR CONTRIBUTIONS

Xiuying Zhao, Kaishou Xu, and Wei Zhou administrated the project. Kaishou Xu and Wei Zhou supervised the study. Xiuying Zhao was a major contributor in writing the manuscript. Xiuying Zhao, Tao Chen, and Binsha Fu analyzed and interpreted the patient data. Zhifu Fu performed visualization of the results. Xiuying Zhao, Tao Chen, Binsha Fu, and Zhifu Fu reviewed and edited the manuscript. All authors read and approved the final manuscript.

FUNDING INFORMATION

This research was funded by Hainan Provincial Health Commission with grant number 21A200163 and Key R & D Plan Project of Hainan Province with grant number ZDYF2021SHFZ091.

CONFLICT OF INTEREST STATEMENT

The authors declare that they have no competing interests.

CONSENT FOR PUBLICATION

Not applicable.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review Board (or Ethics Committee) of Medical Ethics Committee of Hainan General Hospital (protocol code Med‐Eth‐Re [2022] 738 and date of approval November 30, 2022).

Zhao, X. , Chen, T. , Fu, B. , Fu, Z. , Xu, K. , & Zhou, W. (2023). Mutations obstructing ATP's emplacement in KIF2A nucleotide‐binding pocket causes parenchymal malformations, motor developmental delay, with intellectual disability. Molecular Genetics & Genomic Medicine, 11, e2225. 10.1002/mgg3.2225

DATA AVAILABILITY STATEMENT

All data analyzed during this study can be accessed in LOVD database with access number: #0000927493.