Truncated DNM1 variant underlines developmental delay and epileptic encephalopathy

Background Developmental and epileptic encephalopathies (DEEs) signify a group of heterogeneous neurodevelopmental disorder associated with early-onset seizures accompanied by developmental delay, hypotonia, mild to severe intellectual disability, and developmental regression. Variants in the DNM1 gene have been associated with autosomal dominant DEE type 31A and autosomal recessive DEE type 31B. Methods In the current study, a consanguineous Pakistani family consisting of a proband (IV-2) was clinically evaluated and genetically analyzed manifesting in severe neurodevelopmental phenotypes. WES followed by Sanger sequencing was performed to identify the disease-causing variant. Furthermore, 3D protein modeling and dynamic simulation of wild-type and mutant proteins along with reverse transcriptase (RT)–based mRNA expression were checked using standard methods. Results Data analysis of WES revealed a novel homozygous non-sense variant (c.1402G>T; p. Glu468*) in exon 11 of the DNM1 gene that was predicted as pathogenic class I. Variants in the DNM1 gene have been associated with DEE types 31A and B. Different bioinformatics prediction tools and American College of Medical Genetics guidelines were used to verify the identified variant. Sanger sequencing was used to validate the disease-causing variant. Our approach validated the pathogenesis of the variant as a cause of heterogeneous neurodevelopmental disorders. In addition, 3D protein modeling showed that the mutant protein would lose most of the amino acids and might not perform the proper function if the surveillance non-sense-mediated decay mechanism was skipped. Molecular dynamics analysis showed varied trajectories of wild-type and mutant DNM1 proteins in terms of root mean square deviation, root mean square fluctuation and radius of gyration. Similarly, RT-qPCR revealed a substantial reduction of the DNM1 gene in the index patient. Conclusion Our finding further confirms the association of homozygous, loss-of-function variants in DNM1 associated with DEE type 31B. The study expands the genotypic and phenotypic spectrum of pathogenic DNM1 variants related to DNM1-associated pathogenesis.


Introduction
Developmental and epileptic encephalopathy (DEE) are a group of genetic conditions characterized by severe epilepsy affecting children, resulting in delayed development, neurological and non-neurological comorbidities, and, in rare cases, early mortality (1)(2)(3).More than 100 monogenic causes of epilepsies and neurodevelopmental abnormalities have been identified, and genetic factors have since been recognized as significant contributors to DEEs (4,5).
Majority of the de novo deleterious variants occur in the only two GTPase and middle domains manifested in patients with epileptic encephalopathies (11,16,17).Only one deleterious variant c.1603A>G (p.Lys535Glu) has been observed in the PH domain in twin siblings positive for intellectual disability (ID) and autistic symptoms but no epileptic encephalopathy (18).
Neurological abnormalities are a highly heterogeneous group of rare genetic disorders that are primarily diagnosed using whole exome sequencing (WES) (19).Here, we describe a patient harboring a novel de novo non-sense pathogenic variant in the middle domain of the DNM1 protein, whose phenotypes included moderate ID, with mild generalized epilepsy with onset in adolescence.

Ethics statement and study approval
The study presented here describes a proband (IV-2) suffering from a rare neurodevelopmental disorder.The family of the proband showing neurodevelopmental phenotypes was recruited from a remote region of Khyber Pakhtunkhwa (KP), Pakistan.Before the study, the parents were interviewed and patient history was obtained (Figure 1).Informed consent in written form was obtained from the patients and other family members for the publication of the data, results, molecular findings, X-rays, and other related data in this article in compliance with the Helsinki Declaration.Blood samples were collected in Ethylenediaminetetraacetic acid (EDTA) tubes.DNA from blood samples was extracted and quantified using standard methods described previously (20).

Molecular analysis Chromosomal and cytogenetic analysis
Chromosomal and cytogenetic analysis was performed using standard methods as described previously (21).

Whole exome sequencing
WES was performed using DNA from the affected member (II-I).WES and variants filtering steps were performed as described earlier (22, 23), using the Illumina Hi-seq-2500 platform following the manufacturer instructions.After the standard reaction, The data was obtained and evaluated using the bioinformatics procedure into the variant call format (VCF).The VCF was then uploaded into the professional data analysis software (https://basespace.illumina.com/)to filter out the variants.Standard screening principles were used to search for different functional variants.

Determination of variant pathogenicity
The identified variant was first searched in general population databases such as gnomAD (24) and an in-house database to rule out the occurrence of polymorphism.Second, using different online tools, the disease-causing nature of the variant was verified with tools such as MutationTaster (25), DANN (26), LRT (27), FATHMM (28), EIGEN (29), and BayesDel_addAF (30) along with American College of Medical Genetics (ACMG) guidelines (31).

Variant conservation
The conservation of the variant across different species was checked using the NCBI-HOMOLOGENE database (https://www.ncbinlm.nih.gov/homologene).

Sanger sequencing
The variant obtained was filtered and identified as disease causing using Sanger sequencing following standard methods (21).Genomic sequences of the gene were retrieved from the UCSC genome database browser (http://genome.ucsc.edu/cgi-bin/hgGateway).The primers for Sanger sequencing were designed using the PRIMER3 software (32).The exon 11 containing the identified DNM1 variant was amplified using exon-specific primers through polymerase chain reaction (PCR) following standard protocols.Purification of the PCR-amplified DNA was performed according to the manufacturer's instructions.The data obtained from the Sanger sequencing were analyzed using BIOEDIT tool.

In silico analysis-3D protein structure modeling
The crystal structure of the DNM1 protein was retrieved from RCSB under accession number 4UUK.The preparation module of UCSF Chimera (https://www.cgl.ucsf.edu/chimera/)was used to resolve the missing residues and non-sense mutation was inserted using the mutagenesis tool PyMOL, followed by energy minimization with MMFF94 force field implemented in a molecular operating environment (MOE) (https://www.chemcomp.com/Products.htm).The mutation was induced in the structure using the mutagenesis tool implemented in PyMOL (https://pymol.org/2/)as described by Alotaibi et al. (33).

Modeling and structure minimizations
The crystal structure containing missing loop residues at the N terminal, middle, and C terminal were built by the loop module of Chimera; afterward, the structure was subjected to energy minimization.Then, non-sense mutation was inserted at position 468.Surprisingly upon visual inspections and comparison with the 3D structure of wild-type (WT) DNM1, we found that the novel variant undergoes structural changes in the loop regions of the respective protein.

Molecular dynamics simulation
Molecular dynamics simulation was carried out for the wildtype and mutant proteins to comprehend the dynamic behavior of the mutated and wild states.The protein force field ff14SB implemented in the Amber 20 package was applied (34).To solvate each system, the tip3p water model with an 8.0 box dimension was used.Each system was completely solvated and neutralized by the addition of four Na+ ions, which balanced out the charges in each system.After 6,000 steps of the steepest descent and conjugate gradient minimization technique, the density was then equilibrated for 2 ns.For another 2 ns, the entire system was then equilibrated at constant pressure.The temperature was kept constant at 300 K using the Langevin thermostat.Moreover, a 200 ns molecular dynamics (MD) was run after equilibration (33).For electrostatic interactions, the particle Mesh Ewald (PME) method was used.Hydrogencontaining covalent bonds were handled via the SHAKE algorithm.Finally, at constant pressure and temperature MD simulation was performed using PMEMD (35).

RT-qPCR
In order to functionally validate the variant, total RNA was extracted from all the available family members to investigate the relative mRNA DNM1 expression using GAPDH [DQ403057] as the internal control "house-keeping" gene.cDNA was synthesized using standard methods from total RNA using standard cDNA reverse transcription kit (36).Primerbank database (https://pga.mgh.harvard.edu/Parabiosys/)was used to design the primer pair and will be provided on request.PCR SYBRGreen Master Mix was used for the qPCR reaction and Quant-Studio 6 Flex Real-Time PCR-System was used.All the reactions were repeated independently, performed in triplicate, and data were analyzed using Expression-Suite software version 1. GAPDH was used as an endogenous control.

Clinical report
The current study reports on a single affected individual (II-1).Parents were first-degree cousins, and the pedigree depicted an autosomal recessive pattern of inheritance (Figure 1).The family had no previous history of such disorder.The parents and two other siblings were normal.The proband revealed features such as neurodevelopmental disorder, mild microcephaly, moderate to severe ID, speech issues, seizures, epileptic encephalopathy, and hypotonia.
His growth parameters at age 9 were as follows: height: 120.6 cm [2 percentile (−2.1 SD)], weight: 22.7 kg [5 percentile (−1.68 SD)], and head circumference (HC): 48.9 cm [<1 percentile (−2.8 SD)].All the biochemical tests were unremarkable, and no metabolic disorder was observed.Ophthalmological examination showed unremarkable results, including normal funduscopic examination.Electroencephalogram (EEG) revealed multifocal epileptic discharges, thus suggesting epileptic encephalopathy.There were no dysmorphic features observed in the affected individual.He was unable to hold his head without support, roll over, and hold objects properly.Socially, he is shy and avoids making eye contact.Speech and language development were also delayed.The detailed clinical information is mentioned in Table 1.

Brain MRI
The brain MRI revealed no territory infarction, intra cranial hemorrhage, mass effect, hydrocephalus, or midline shift.A generalized volume loss was observed manifested by a prominent ventricles and extra-axial cerebrospinal fluid (CSF) spaces (Figure 2).Posterior fossa structures showed unremarkable presentation.Paranasal sinus, mastoid air cells, and visualized orbits were unremarkable.A delay in myelination of the midline structures was observed, corresponding to the patient's age.No abnormality associated with parenchymal signal intensity was identified.No extra-axial collections, hydrocephalus, and/or spaceoccupying lesions were observed.The posterior fossa structures, intracranial arteries, and veins were unremarkable.The paranasal sinuses and visualized parts of the orbits were unremarkable.

Molecular genetic analysis
Chromosomal and cytogenetic analysis did not reveal any disease-causing genomic alterations that might be the cause of the observed phenotype in the proband.The DNA of the affected individual (II-2) was subjected to WES using standard methods.WES filtration data analysis revealed a novel homozygous nonsense mutation (c.1402G>T; p.Glu468*) in the exon 11 of the DNM1 (NM_004408.4;9q34.11;ENST000003729) gene.The novelty of the variant was checked in the online literature and HGMD database 2022.The variant results in a premature stopcodon at 468 amino acid position.The mutation might result in a shorter protein that might result in non-sense-mediated decay (NMD).

Sanger sequencing
To consider the segregation of the variant with the disease phenotypes, the identified variant [c.1402G>T] was Sanger sequenced in all family members, which segregated perfectly within the family.The variant was observed in homozygous state in the proband, both the parents (III-2, III-3) and sibling (brother; IV-3) were carriers, and the sibling (brother; IV-1) was wild type (Figures 3A-C).

Pathogenicity and conservation
The identified non-sense variant (p.Glu468*) was classified as disease causing using different online prediction tools such as MutationTaster (disease-causing [1]), DANN (disease-causing [0.997]), LRT (pathogenic [0]), FATHMM (pathogenic [0.9844]), EIGEN (pathogenic [1.130]), and BayesDel_addAF (pathogenic [0.5774]), and according to the ACMG guidelines, the identified variant was classified as pathogenic (Class I).According to gnomAD, the variant was not observed in homozygous state in the public database thus suggesting a non-polymorphic nature of the variant.Furthermore, the Glu amino acid at position 468 is highly conserved across several species (Figures 4A,B), suggesting key role of this variant in the neurodevelopment.

In silico analysis-3D protein modeling
Then non-sense mutation was inserted at position 468 as shown in Figure 4A.Remarkably upon visual inspections and comparison with the 3D structure of wild-type DNM1, we found that the novel variant undergoes structural changes in loop regions of the respective proteins.If the DNM1 protein is not degraded in the NMD, these changes in the final protein structure might lead to a small non-functional protein that might not perform proper function and downstream signaling (Figures 5A,B).

RMSD analysis
Root mean square deviation (RMSD) analysis showed that the value of mutant increased rapidly to 4.5 Å from 10 to 15 ns but then decreased to 2 Å for 50 ns.After 50 ns, RMSD slightly increased to 2.5 Å and minor deviations were seen during 65-68 and 85-90 ns, but overall, the structure showed a stable behavior till 100 ns MD run.On the other hand, the wild-type DNM1 revealed a stable behavior during the entire 100 ns MD simulation, only minor deviations were seen during 55-75 ns and the DNM1 Wt protein RMSD value of 2.8 Å was attained, and throughout the simulation, it revealed stability.RMSD plots of both the wild-type and the mutated proteins are shown in Figure 6A.

Rg analysis
By analyzing the structural compactness along with the stability of the folded or unfolded protein, the radius of gyration (Rg) calculation was carried out.For the mutant and wild states, the average values of Rg were in the range of 29.5-31.0 and 31.0-32.0,respectively.It is clear from the Rg calculation that the wild-type system revealed more compactness as compared to the mutant form during the MD simulation.The Rg plots for both the wild and mutant states are shown in Figure 6B.

RMSF analysis
The root mean square fluctuation (RMSF) was calculated for the C-alpha atoms of both the wild and mutant states in order to explore the fluctuating residues during the 100 ns MD simulation.In the mutant state, the residues 105-107, 110-113, and residue 325 showed more fluctuations during the MD simulation while the residues 114-324 showed stability.On the other hand, the wild state showed major fluctuations at residues 648-652 and the residues 740-750 showed minor fluctuations, while all other residues showed stability during the entire 100 ns MD run. Figure 6C displays the RMSF plots for the wild-type as well as mutant protein.

Non-sense variant in DNM1 reduced mRNA expression
The relative expression data of DNM1 gene in the affected individual, parents, and normal control individuals showed that the proband (IV-2) having the disease-causing homozygous variant (p.Glu468*) had substantial reduction in the DNM1 gene expression as compared to the wild type (control) and carrier (parents) (Figure 7).
DNM1 gene encodes dynamin-1, a protein required for clathrinmediated endocytosis and synaptic vesicle recycling.The fission of clathrin-coated vesicles from the plasma membrane by dynamin-1 oligomers promotes vesicle-mediated neurotransmitter release at the synapse (14).It is only found in neurons in the presynaptic terminal, where it participates in synaptic vesicle endocytosis and membrane recycling after neurotransmitter release (14).In mammals, this gene can produce various isoforms, which are characterized primarily by alternative splicing of the tenth exonsome transcripts have exon 10b, while others contain exon 10c (42).
Herein, we describe a proband having pathogenic Class-I variant in the DNM1 that revealed neurodevelopmental features such as developmental delay, moderate-severe ID, microcephaly, hypotonia, seizures, and speech issues.Features reported previously, such as visual impairment, EEG abnormality, and scoliosis, were not observed in the patient reported here (Table 1).Interestingly, Yigit et al. (41) reported the only two affected individuals having homozygous DNM1 variants [c.97C>T; p.(Gln33*), c.850C>T; p.(Gln284*)] that were having a non-sense mutation and revealed hypotonia, spasticity, dystonia, and feeding abnormalities that overlapped with our patient features (41).However, scoliosis and visual impairment were not observed in our patient.
Previously, only de novo variants in the DNM1 were linked to developmental and epileptic encephalopathy (Table 2; MIM 620352, MIM 616346).Clinically, affected individuals having DNM1 heterozygous missense variants show phenotypic heterogeneity with early-onset epilepsy, hypotonia, and developmental delay (11).These patients have normal MRI analysis with cerebral volume loss over time.Five patients with early-onset DEE (DEE31, OMIM 616246) were the first to have heterozygous missense mutations in the DNM1 gene (13).Since then, many patients having heterozygous, primarily de novo DNM1 mutations, have been reported in the literature (Table 2).Except for one de novo in-frame 06 bp insertion, all observed variants disrupt highly conserved residues and are projected to have a dominant-negative impact on the dynamin-1 function.Detailed clinical comparison of all the patients reported with DNM1 variants have been summarized in Table 3.
To date, only 69 variants have been reported in the DNM1 gene including two splice-site variants, three small deletions, three small insertions, seven non-sense, and 54 missense variants (Table 2).All missense variants associated with epileptic encephalopathy cluster in the two major functional domains of the DNM1 protein, the GTPase domain, and the middle domain.In addition, homozygous loss-of-function pathogenic variants in DNM1 have been reported to cause a severe autosomal recessive developmental and epileptic encephalopathy in three unrelated patients (37,41).Recurrent DNM1 de novo splice-site variant has also been associated with developmental and epileptic encephalopathy with a dominantnegative mechanism (44).The p.Arg237Trp mutation has been observed in approximately one-third of patients.The nonsense variant identified in the present study affects a highly conserved Glu residue within the middle domain that plays a vital role in binding different targets and helps in DNM1 multimerization.
Molecular dynamics simulation is a useful tool to assess the genuine behaviors of biological molecules and their surroundings (19).In the present study, we performed MD simulations on two systems, native and mutant DNM1 p.Glu468* .We considered parameters such as RMSD, RMSF, and Rg between the native and mutant p.Glu468* DNM1 protein structures.In terms of RMSD, DNM1 p.Glu468* after initial increase and decrease showed stable behavior till 100 ns MD run while DNM1 WT revealed stable behavior during the entire 100 ns MD simulation except for some minor deviations at 55-75 ns of MD run.The RMSF trajectory for DNM1 p. Glu468* showed fluctuations at the residues 105-107, 110-113, and 325 while the residues 114-324 showed stability along with native DNM1 that showed major and minor fluctuations at residues 648-652 and 740-750, respectively.Rg denotes the overall spread of the molecule, the average values of Rg were indicated in the range of 29.5-31.0Å and 31.0-32.0Å for DNM1 p. Glu468* and wild type, respectively.It is clear from the Rg calculation that the wild-type system revealed more compactness as compared to the mutant form during the MD simulation.Overall, an MD run demonstrated that the non-sense variant in DNM1 protein leads to instability of the p. Glu468* system in comparison to the wild-type protein as manifested by varied trajectories of RMSD, RMSF, and

Somatic growth
Weight at follow-up n.r.Rg, respectively.In addition, functional validation of this non-sense variant in the DNM1 gene using RT-qPCR approach revealed a decreased expression of the gene in the index patient signifying loss-of-function effect (45).For families impacted by rare inheritable diseases, proper genetic counseling is crucial.Additionally, prenatal genetic screening/diagnosis is the most effective management method for these medical conditions, for which there is no cure at this time (46).In summary, we reported a novel non-sense variant in the DNM1 leading to severe neurodevelopmental phenotype in the patient that includes early-onset epilepsy, hypotonia, intellectual disability, seizures episodes, and pronounced developmental delay.This study will aid in genetic counseling and bringing awareness regarding the disease in the common population.The study also adds to the variant spectrum of DNM1 and contributes to the evaluation of genotype-phenotype correlation.

FIGURE 1 Four
FIGURE 1Four-generation pedigree of a family in the present study showing consanguinity (double lines) and autosomal recessive inheritance of the disease.The patient is highlighted with a black square, while an asterisk sign represents individuals subjected for WES.Slashes represent diseased individuals.

FIGURE 2 Brain
FIGURE 2Brain MRI of affected individual (IV-2) revealed generalized volume loss manifested by a prominent ventricles and extra-axial CSF spaces.Posterior fossa structures, paranasal sinus, mastoid air cells, and visualized orbits were unremarkable.

FIGURE 5
FIGURE 5Representation of the 3D structural model of human wild-type DNM1(wt)  protein (pink) and mutated protein (light blue).Red color indicates position of novel mutation Glu at position 468 in DNM1 p.Glu468* protein identified in this study.Accession number NP_001275668.1 version was used.

FIGURE 7 RT
FIGURE 7RT-qPCR shows a significant decrease of the DNM1 mRNA expression in an index patient (IV-2) in comparison to control or other members of the family (III-2, III-3, IV-1, and IV-3).

TABLE 1
Clinical characteristic and general information of a proband with diverse neurodevelopmental phenotypes.

TABLE 2
Mutations reported in DNM1 associated with neurodevelopmental disorders.