The patient, a 3-year-old boy, was born at term through an uneventful delivery with normal birth parameters of consanguineous Chinese parents. He was the first child in a second pregnancy, after the first resulted in an induced abortion due to fetal death. The grandparents of the patients were cousins. The pedigree of the family was as follows (Figure 1A). The age at the first visit was 5 months. The first symptoms observed were motor and cognitive retardation. He could not attain head control, turn over, or actively grasp objects. He had normal eyesight and hearing. The second symptom, paroxysmal hypertonia, began as early as 1 month after birth. Initially, the symptoms were clenched fists with both hands and straightened limbs, which gradually progressed to flexion of both upper limbs, fisted hands, and straightened lower limbs. During the course, consciousness was clear and the symptoms lasted for a few seconds to a few minutes before spontaneous relief. Physical examination revealed microcephaly, white skin, yellow hair, slightly long face, high palatine arch, right hand penetration, small penis, and small hands and feet. His muscle force was grade three, and he had paroxysmal hypertonia, negative limb deep reflection, and bilateral symmetric flexor plantar reflex.
Magnetic resonance imaging (MRI) showed ventricular broadening and left lateral fissure widening in 2017. 1 year later, MRI revealed symmetric hyperintensity in the bilateral basal ganglia on T2-weighted imaging, T2-FLAIR, and DWI sequences (Figure 1B). Video electroencephalogram showed a normal background and recorded paroxysmal limb rigidity with no epileptiform discharges. Liver and renal function, myocardial enzyme, and ceruloplasmin levels were normal, but metabolism screening showed mild ketonuria and hyperlactacidemia (2.55-3.36). G-banding karyotype analysis: 46, XY. Gesell Developmental Scale: Adaptability: DQ 26.7, DA 2.5, severe delay; Gross motor: DQ 23.7, DA 2.2, extremely severe delay; Fine motor: DQ 24.7, DA 2.3, extremely severe delay; Communication: DQ 49.5, DA 4.6, moderately delayed; personal-social skills: DQ 37.6, DA 3.5, severe delay.
DNA was extracted from peripheral blood using the QIAamp DNA Blood Mini Kit (QIAGEN). A total of 3 mug of patient DNA was sheared to produce 200-300 base pair (bp) fragments. Whole-exome enrichment was performed using an Agilent SureSelect Target Enrichment system (Agilent Technologies). Coding exons and flanking intronic regions were enriched with the Agilent SureSelect Human All Exon V6 reagent (Agilent Technologies), according to the manufacturer's protocol. Captured libraries were loaded onto a HiSeq 2500 platform (Illumina). Base calling and assessments of sequence read quality were performed using Illumina Sequence Control Software (SCS; Illumina), which provided real-time analyses. Reads with average quality scores <25 were removed, and bases with quality scores <20 were trimmed. The mean read depth of each sample was 100 x. The reads were aligned to the human reference genome (UCSC GRCh37/hg19) using the Burrows-Wheeler Aligner (BWA v0.7.15) BWA-MEM algorithm. Reads with low mapping quality scores were removed. Presumed PCR duplicates were identified using Picard's MarkDuplicates and removed. Local alignment optimization and base quality recalibration were performed using the Genome Analysis Toolkit (GATK). Single-nucleotide variants (SNVs) and InDels were identified with Mutect2, respectively, and saved in a variant call format. Variants were functionally annotated and filtered with ANNOVAR (http://annovar.openbioinformatics.org/en/latest), which provided built-in public databases (OMIM, InterVar, ClinVar, HGMD, Cosmic70, dbSNP, 1000G, ESP, ExAC, and gnomAD), and the HGMD Professional database. We identified a de novo variant (c.116 G>A, p.S39N) in exon 2 of DNM1L, which has not been reported previously. Furthermore, gene variation was validated using Sanger sequencing (Figure 1C). The conservation of the identified amino acid across different species and the protein 3D modeling for the DNM1L-wild and identified missense variant were shown in Figures 1D-F. The pathogenicity score was checked using the following online tools (Table 1).
Benzhexol (0.5 mg BID) and Medopar (31.25 mg QID) were used to reduce muscle tension, and a cocktail therapy (VitB1 10 mg TID, VitC 0.5 g BID, VitE 100 mg QD, CoQ 10 mg TID and L-carnitine 100 mg BID) was administered to treat the primary illness, but none provided significant clinical benefit. At the last follow-up, the patient was 3 years and 4 months of age. He was 85 cm tall and weighed 8 kg, with a head circumference of 46 cm. He was admitted to our hospital with symptoms including spastic tetraparesis, no postural control, paroxysmal hypermyotonia and tremor, and severe cognitive impairment but no seizures. He could only follow a voice and light, occasionally respond to communication, and make vowel sounds, but he could not cry or laugh out loud.
We searched for reported cases in the PubMed database using the terms "DNM1L" and "mitochondrial." Thirty-five cases from 20 relevant references were retrieved; these included the first case reported by Waterham in 2007 and other cases reported from January 2015 to date. A total of 36 cases were analyzed together with the case reported by us. Fifteen males, 16 females, and two patients without reference to sex and three pedigrees (both male and female) were included. The median age of onset was 6 months (range, neonatal period: 9 years).
Clinical manifestations included psychomotor retardation in 77.8% (28/36) of patients, limb paralysis in 66.7% (18/27), dystonia in 82.8% (24/29), and epilepsy in 59.4% (19/32). Other symptoms included ataxia in six cases, nystagmus in three cases, optic atrophy in six cases, dysarthria in four cases, microcephaly in four cases, pain insensitivity in two cases, and sensory and motor axonal neuropathy in two cases.
On auxiliary examination, 83.3% (25/30) of the cases had abnormalities in brain MR images, including abnormal hyperintensity in the cortex (10 cases), basal ganglia/thalamus (10 cases), subcortical white matter (three cases), brain atrophy (12 cases), lactate peak (four cases), and thinning/absence of the corpus callosum (three cases). Seventy-five percent (15/20) of the cases showed abnormalities in EEG, such as epileptic discharge or a weak background. Hyperlactacidemia was identified in 60.7% (17/28) of the cases, and decreased respiratory chain enzyme activity was detected in 28.6% (4/14) of cases. Muscle biopsy was performed in 22 cases, ragged red fibers were observed in four cases, and long tangled or swollen mitochondria were observed in 12 cases.
At the last follow-up, the 23 surviving patients had a median age of 7 years (2-27 years), whilst 12 had died with a median age at death of 1.5 years (from the neonatal period to 13 years). The median course of follow-up was 3.5 years (range, 7-24 years), and the median course of disease from illness to death was 1 year (range, 8-11.5 years). At the last follow-up, barring three families with optic atrophy as an isolated symptom, the rest had varying degrees of dyskinesia (four patients could walk with assistance, two could stand with assistance, one could sit alone, 11 could not control body position, and the rest were not mentioned), cognitive impairment, dystonia, or epileptic seizure.
De novo variants were identified in 72.2% (26/36) of the cases, 13.9% (5/36) had heterozygous dominant variants, 11.1% (4/36) had compound heterozygous variants, and 2.8% (1/36) had homozygous variants. A total of 24 DNM1L variants were identified, including 21 missense variants, two deletion variants, and one duplication variant (Table 2).
The clinical manifestations of the GTPase domain group were milder than those of the middle domain group (Figure 1G, Table 3). The prevalence of psychomotor retardation and epilepsy in the GTPase domain group was lower than that in the middle-domain group (Psychomotor retardation: GTPase domain group, 50.0% [6/12] vs. middle domain group, 91.3% [21/23]; epilepsy: GTPase domain group, 8.3% [1/12] vs. middle domain group, 89.5% [17/19]); these differences were statistically significant (P < 0.05). In the five patients with heterozygous variants, three pedigrees with variants in the GTPase domain presented with optic atrophy as the isolated symptom, which was the mildest phenotype, and the family pedigrees were shown together with the segregation of the DNM1L variants in the affected individuals. The variants of the two other patients were located in the middle domain; one patient had a c.1048G>A, p.G350R variant, and variant analysis by Sanger sequencing suggested a low level (6-8%) of maternal mosaicism. The patient presented with developmental delay, hypotonia, status epilepticus, and elevated serum lactate levels, and he died of status epilepticus at the age of 5. Another patient had two heterozygous DNM1L variants (c.1085G>A, p.Gly362Asp; c.1535T>C, p.lle512Thr) on the same allele (maternal allele). His mother with the variant (c.1535T>C, p.lle512Thr) was healthy, but the other variant (c.1085G>A, p.Gly362Asp) was not present in either parent. However, further analysis of the DNM1L gene using a next-generation sequencing approach revealed that the variant (c.1085G>A, p.Gly362Asp) was present in the mother's blood DNA at a very low level. Furthermore, the patient's half-brother had died at the age of 3 years. Therefore, these findings supported maternal germline mosaicism for a dominant variant (c.1085G>A, p.Gly362Asp). The patient manifested developmental delay prior to the onset of illness. At 5 years and 5 months of age, he presented with partial motor status epilepticus after a viral illness. 4 months later, he presented with spastic quadriplegia and hyperkinesis but poor voluntary movements, no postural control, and severe cognitive impairment. However, there was no statistically significant difference in the survival rate in the different domain groups (log-rank test, P = 0.39, Figure 1H).
Auxiliary examination revealed that 83.3% (25/30) of the cases had abnormalities in brain MR images; 62.5% (5/8) of the GTPase domain group vs. 90.9% (20/22) of the middle domain group (P = 0.102), with no statistically significant difference. Seventy-five percent (15/20) of the cases showed abnormality in EEG; 20.0% (1/5) of the GTPase domain group vs. 92.9% (13/14) of the middle domain group (P = 0.006), with statistically significant differences.