Introduction
Microcephaly with or without chorioretinopathy, lymphedema, or mental retardation (MCLMR; MIM# 152950) is clinically characterized by a broad spectrum of symptoms, including central nervous system involvement, which manifests as mild to severe microcephaly and congenital optical anomalies, especially chorioretinopathy, lower-limb lymphedema, and several characteristic facial features1. Moreover, some patients develop mild to moderate mental retardation2. The prevalence of this rare neonatal disorder is estimated at fewer than 1 per 1,000,000 live births. Furthermore, it is inherited in an autosomal dominant pattern with variable expressions and reduced penetrance3. A mutation in the kinesin family member 11 (KIF11) gene has been recently described as the primary cause of the disease4.
The KIF11 gene (MIM# 148760), also known as EG5; HKSP; KNSL1; MCLMR; TRIP5, is located on 10q23.33. This gene spans 62,266 bases of the genomic sequence, encompassing 22 coding exons and encodes a homotetrameric kinesin motor complex with 1056 residues. The members of this protein family are involved in various microtubule activities, mostly during cell mitosis, which encompasses centrosome separation, chromosome positioning, and the establishment of a bipolar spindle at the time of cell mitosis. Furthermore, in non-mitotic cells, this protein is required for the transportation of secretory proteins from the Golgi apparatus to the cell membrane5, 6.
Next-generation sequencing (NGS) technologies are widely used in various fields of pediatric disorders to better understand cases with unknown origins, identify unexpected clinical manifestations, and recognize complications that may occur in the clinical course of the disease. These technologies have significant advantages in term of predicting disease and symptoms. Also, the association of alterations with genes already known to be pathogenic, the extension of the clinical manifestations of known diseases, the determination of prognosis, and the identification of new genes whose mutations cause disease are among the uses of this technique7.
In the present study, we reported a novel heterozyeous missense mutation in an Iranian family with MCLMR. It is identified by the whole-exome sequencing (WES) approach and segregated with direct sequencing of the coding region of the KIF11 gene. Also, we updated the literature by comparing our patient's features with those of the other patients reported previously and provided future viewpoints for clinical study in this area. Our findings broaden both the understanding of the clinical phenotype and the allelic repertoire of KIF11.
Results
Clinical Presentation
The proband was born underweight through vaginal delivery at 33 weeks gestation in the first pregnancy. Her birth weight, length, and head circumference were measured at 1.250 kg, 43 cm, and 26 cm, respectively.
After birth, she had a low head circumference and was slightly underweight but did not have any other problems. Up to 16 months, she experienced delays in sitting and walking, and she started walking at the age of 16 months. At the age of 3 years, she was admitted to the paediatrician for poor hearing and eyesight. The results of the audiological evaluation, using OAE (Otoacoustic Emission), ABR (Auditory Brainstem Response), and ASSR (Auditory Steady-State Response), showed that she was sedated with chloral hydrate. Moreover, the audiological evaluation revealed that TEOAE responses were abnormal in both ears. Additionally, ABR wave V in the right and left ears was traced down to 70 Db and 90 Db NHL, respectively, and ASSR threshold measurement indicated moderate to severe hearing loss in both ears. Besides, MRI evaluation showed that the cerebral hemispheres were smaller than normal. Likewise, thick irregular cortical gyri in both parietal and occipital regions were observed. Furthermore, eye examination revealed that the right and left eye prescriptions were +8 and +9, respectively. For poor eyesight, the doctor prescribed appropriate eyeglasses, and for managing the poor hearing, cochlear implantation was performed. Also, no chromosomal aberrations were detected in the cytogenetic analysis (46,XX).
Based on the mentioned signs, such as developmental delay, microcephaly, small face, low-set ears, hearing loss, visual defects, and seizures, some inconclusive clinical diagnoses were given (Figure 1).
Identification of a Novel Missense <italic id="e-ea531ef0da56">KIF11 </italic>Variant
WES detected 118,824 variants, of which 24,530 variants were located on the exonic or exonic-splicing region. Variant filtering was conducted based on ACMG guidelines, leaving 1,322 variants for further analysis. The details of the filtering strategy are summarized in Table 1. Given the autosomal dominant inheritance, a likely pathogenic missense mutation (c.629T>C) in the KIF11 gene was discovered at amino acid position 210 (p.Val210Ala). The variant was not documented in population variant databases, including ClinVar, 1000 Genomes (October 2015), dbSNP, Exome Aggregation Consortium (ExAC), Cambridge, and IRANOME.
The results of Sanger sequencing validated that this variant is well segregated with the disease, as the variant was not found in other healthy family members, including parents and grandparents (Figure 2).
<italic id="e-020b6b6a2faf">In Silico</italic> Protein Analysis
According to BLAST, residues of 16-368 belong to the Kinesin motor domain, BimC/Eg5 spindle pole proteins (accession: cd01364), which participate in spindle assembly and chromosome segregation during cell division (Figure 3A).The 6TA3 Protein Data Bank (PDB) record was detected for the human kinesin-5 motor domain in the GSK-1 state bound to microtubules. The microtubules and all non-protein atoms were removed to attain the kinesin-like protein KIF11 structure using MOE software. Figure 4A shows this protein structure before and after monomerization.
ConSurf and Interprosurf results determine functional sites on the protein structure surfaces. These results show that residues 12, 16, 169, 171, 172, 175, 176, 179, 183, 203, 204, 206, 207, 210, 211, 212, 214, 220, 221, 223, 224, 225, 227, 228, 231, 302, 303, 304, and 327 are the most functional sites in the protein structure (Figure 4B). Using SIFT, the V210A amino acid substitution was predicted as a “tolerated substitution”. However, consistent with PolyPhen-2, PROVEAN analyzer, and PHD-SNPg results, this mutation was predicted to be probably deleterious and damaging. Furthermore, PolyPhen-2 displays a multiple sequence alignment for 75 amino acid residues surrounding the variant’s position in the query sequence (Figure 3B). Moreover, I-Mutant2.0 predicted a decrease in the stability of the mutation.
According to HOPE results, the mutated residue lies in a domain that is vital for the binding of other molecules and is also in contact with residues in another domain that is crucial for binding. The interplay of these two domains could be disturbed by the mutation, exerting an adverse effect on protein function by disrupting the signal transfer from the binding domain to the activity domain (Figure 5).
In addition, based on data obtained from a known database and the estimation of protein-protein interactions, STRING (https://string-db.org/) demonstrated that this protein has several functional partners, which mostly belong to the TRAFAC class myosin-kinesin ATPase superfamily, and are involved in cell cycle cytokinesis through Rho-mediated signaling, chromosome congression, microtubule-kinetochore conjugation, spindle assembly checkpoint activation, and other key processes in cell division and cell cycle progression.
Discussion
MCLMR, a rare autosomal dominant or sporadic condition, is a congenital malformation characterized by variable expression of microcephaly, eye disorders such as chorioretinopathy, congenital lymphedema, and mild-to-moderate intellectual disability. Additionally, the most commonly reported cause of this disorder is KIF11 mutations.
In this study, we describe an Iranian girl from a non-consanguineous marriage with primary microcephaly and a novel heterozygous missense mutation in KIF11 (p.Val210Ala). KIF11, an evolutionarily conserved microtubule-associated protein, is involved in mitotic spindle dynamics, centrosome separation, and microtubule-based movements of vesicles and other organelles in non-dividing cells10, 11. KIF11 protein knockdown, in any form, results in monopolar spindle formation, defects in centrosome separation, and metaphase arrest12.
To date, 67 cases have been described with KIF11 mutations and MCLMR2, 3, 13, 14, 15, 16, 17, 18, 19, 20. The majority of the currently described cases were missense (8/67), splicing (12/67), nonsense (14/67), frameshift (24/67), and deletion (2/67) (Table 2). It demonstrated that aside from point mutations, KIF11 haploinsufficiency caused by deletion is linked to autosomal dominant microcephaly, chorioretinopathy, and mild intellectual disability. In all of them, the missense mutations affected highly conserved residues, or a prematurely truncated protein was formed because of a frameshift mutation. Also, among different reports, most of the mutations were sporadic (1/67), heterozyous (18/67), and maternal (5/67)2, 13, 14, 15, 16, 17, 18, 19, 20, 21. Moreover, several heterozygous de novo (18/67) mutations in the KIF11 gene in MCLMR patients have been reported, and the variant identified in our study is compatible with their results2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21.
In line with these studies, we detected a heterozygous mutation in KIF11 causing loss of function by affecting the N-terminal domain. This catalytic (head) domain has ATPase activity (between residue 105 and 112) and belongs to the larger group of P-loop NTPases. Additionally, three internal domains in the protein structure contribute to multimerization into a homo-tetramer with coiled-coil linkage22. For kinesin motor domains to move along the microtubule with ATP hydrolysis, kinesin head groups work in pairs. Also, the neck linker binds to the motor domain, which repositions the other head domain through the coiled-coil domain close to a second tubulin dimer. ATP hydrolysis in the kinesin motor domain triggers a conformational change that pulls the first domain forward23, 24.
Moreover, the mutation found is located in a protein α-helix domain in buried regions that are important for the binding of other domains. The wild-type and mutant amino acids differ in size, with the mutant residue being smaller than the wild-type, and the mutation will cause space in the core of the protein. Therefore, the amino acid alteration in this region interferes with protein binding and domain-domain interactions. As this motor protein is very conserved and vital in many species, any structural changes result in a wide range of disturbances. In addition, the pathogenicity degree demonstrated in different predictive ways indicates the deleteriousness of this mutation, and it is compatible with our patient's clinical signs.
Based on the clinical presentations among MCLMR patients, the most prevalent craniofacial features were a broad nose with a rounded tip (26/67), a long philtrum and prominent chin (25/67), prominent ears (24/67), and up-slanting palpebral fissures (24/67) (Table 3). Our case also had different craniofacial attributes related to MCLMR, including a broad nose with a rounded tip, prominent ears, a small face, low-set ears, and up-slanting palpebral fissures. Likewise, among the patients with KIF11 mutations and MCLMR, eye problems, specifically chorioretinopathy, were more prevalent (56/67). Comparatively, our proband showed poor eyesight. Among different CNS disorders, ventricular stenosis (3/67), a small frontal lobe (7/67), intellectual disability (24/67), learning problems (34/67), and behavioral problems (11/67) were common (Table 3). Additionally, microcephaly was present in all except P59, typically resulting from abnormalities in the regular neurogenesis process, leading to inadequate neuron generation during cortical development25.
Intriguingly, previous studies have shown that several genes such as CENPJ, MCPH1-17, ASPM, CDK5RAP2, STIL, CEP152, and WDR62, which are involved in a recessive pattern of microcephaly, play a role in centrosome formation and spindle development26. These encode centrosomal proteins, essential for various processes like centriole duplication, centrosome maturation, spindle assembly, microtubule dynamics, and regulation of the cell cycle27.
The kinesin motor’s function, in cooperation with microtubules and associated proteins, supports the development of neuronal progenitors, the movement of neurons, and the intracellular transport of neurons and dendrites28.
The results of our study highlighted the important role of molecules involved in the function of mitotic spindles in CNS development by showing the presence of mutations in the KIF11 gene that lead to the dominant form of microcephaly. Moreover, the study on zebrafish revealed that the inhibitor mutation in KIF11 led to a mitotic arrest in radial glia, serving as resident neural stem cells. Thus, the loss of function of KIF11 causes a noticeable reduction in oligodendroglia and neurogenesis29.
Other significant clinical presentations in patients with KIF11 mutations were developmental delay (6/67), hearing impairment (4/67), speech delay (8/67), and cardiac anomaly (3/67) (Table 3). Additionally, in 37 cases, lymphedema was presented. The deleterious effect of the identified missense mutation on the function of KIF11 in this study is compatible with our patient's clinical signs.