RNA sequencing and target long-read sequencing reveal an intronic transposon insertion causing aberrant splicing.

More than half of cases with suspected genetic disorders remain unsolved by genetic analysis using short-read sequencing such as exome sequencing (ES) and genome sequencing (GS). RNA sequencing (RNA-seq) and long-read sequencing (LRS) are useful for interpretation of candidate variants and detection of structural variants containing repeat sequences, respectively. Recently, adaptive sampling on nanopore sequencers enables target LRS more easily. Here, we present a Japanese girl with premature chromatid separation (PCS)/mosaic variegated aneuploidy (MVA) syndrome. ES detected a known pathogenic maternal heterozygous variant (c.1402-5A>G) in intron 10 of BUB1B (NM_001211.6), a known responsive gene for PCS/MVA syndrome with autosomal recessive inheritance. Minigene splicing assay revealed that almost all transcripts from the c.1402-5G allele have mis-splicing with 4-bp insertion. GS could not detect another pathogenic variant, while RNA-seq revealed abnormal reads in intron 2. To extensively explore variants in intron 2, we performed adaptive sampling and identified a paternal 3.0 kb insertion. Consensus sequence of 16 reads spanning the insertion showed that the insertion consists of Alu and SVA elements. Realignment of RNA-seq reads to the new reference sequence containing the insertion revealed that 16 reads have 5' splice site within the insertion and 3' splice site at exon 3, demonstrating causal relationship between the insertion and aberrant splicing. In addition, immunoblotting showed severely diminished BUB1B protein level in patient derived cells. These data suggest that detection of transcriptomic abnormalities by RNA-seq can be a clue for identifying pathogenic variants, and determination of insert sequences is one of merits of LRS.

A novel de novo KCNB1 variant altering channel characteristics in a patient with periventricular heterotopia, abnormal corpus callosum, and mild seizure outcome.

KCNB1 encodes the α-subunit of Kv2.1, the main contributor to neuronal delayed rectifier potassium currents. The subunit consists of six transmembrane α helices (S1-S6), comprising the voltage-sensing domain (S1-S4) and the pore domain (S5-P-S6). Heterozygous KCNB1 pathogenic variants are associated with developmental and epileptic encephalopathy. Here we report an individual who shows the milder phenotype compared to the previously reported cases, including delayed language development, mild intellectual disability, attention deficit hyperactivity disorder, late-onset epilepsy responsive to an antiepileptic drug, elevation of serum creatine kinase, and peripheral axonal neuropathy. On the other hand, his brain MRI showed characteristic findings including periventricular heterotopia, polymicrogyria, and abnormal corpus callosum. Exome sequencing identified a novel de novo KCNB1 variant c.574G>A, p.(Ala192Thr) located in the S1 segment of the voltage-sensing domain. Functional analysis using the whole-cell patch-clamp technique in Neuro2a cells showed that the Ala192Thr mutant reduces both activation and inactivation of the channel at membrane voltages in the range of -50 to -30 mV. Our case could expand the phenotypic spectrum of patients with KCNB1 variants, and suggested that variants located in the S1 segment might be associated with a milder outcome of seizures.

A deep intronic TCTN2 variant activating a cryptic exon predicted by SpliceRover in a patient with Joubert syndrome.

The recent introduction of genome sequencing in genetic analysis has led to the identification of pathogenic variants located in deep introns. Recently, several new tools have emerged to predict the impact of variants on splicing. Here, we present a Japanese boy of Joubert syndrome with biallelic TCTN2 variants. Exome sequencing identified only a heterozygous maternal nonsense TCTN2 variant (NM_024809.5:c.916C >T, p.(Gln306Ter)). Subsequent genome sequencing identified a deep intronic variant (c.1033+423G>A) inherited from his father. The machine learning algorithms SpliceAI, Squirls, and Pangolin were unable to predict alterations in splicing by the c.1033+423G>A variant. SpliceRover, a tool for splice site prediction using FASTA sequence, was able to detect a cryptic exon which was 85-bp away from the variant and within the inverted Alu sequence while SpliceRover scores for these splice sites showed slight increase (donor) or decrease (acceptor) between the reference and mutant sequences. RNA sequencing and RT-PCR using urinary cells confirmed inclusion of the cryptic exon. The patient showed major symptoms of TCTN2-related disorders such as developmental delay, dysmorphic facial features and polydactyly. He also showed uncommon features such as retinal dystrophy, exotropia, abnormal pattern of respiration, and periventricular heterotopia, confirming these as one of features of TCTN2-related disorders. Our study highlights usefulness of genome sequencing and RNA sequencing using urinary cells for molecular diagnosis of genetic disorders and suggests that database of cryptic splice sites predicted in introns by SpliceRover using the reference sequences can be helpful in extracting candidate variants from large numbers of intronic variants in genome sequencing.

Genome sequencing and RNA sequencing of urinary cells reveal an intronic FBN1 variant causing aberrant splicing.

Exome sequencing and panel testing have improved diagnostic yield in genetic analysis by comprehensively detecting pathogenic variants in exonic regions. However, it is important to identify non-exonic pathogenic variants to further improve diagnostic yield. Here, we present a female proband and her father who is diagnosed with Marfan syndrome, a systemic connective tissue disorder caused by pathogenic variants in FBN1. There are also two affected individuals in the siblings of the father, indicating the genetic basis in this family. However, panel testing performed by two institutions reported no causal variants. To further explore the genetic basis of the family, we performed genome sequencing of the proband and RNA sequencing of urinary cells derived from urine samples of the proband and her father because FBN1 is strongly expressed in urinary cells though it is poorly expressed in peripheral blood mononuclear cells. Genome sequencing identified a rare intronic variant (c.5789-15G>A) in intron 47 of FBN1 (NM_000138.4), which was transmitted from her father. RNA sequencing revealed allelic imbalance (monoallelic expression) of FBN1, retention of intron 47, and fewer aberrant transcripts utilizing new acceptor sites within exon 48, which were confirmed by RT-PCR. These results highlighted urinary cells as clinically accessible tissues for RNA sequencing if disease-causing genes are not sufficiently expressed in the blood, and the usefulness of multi-omics analysis for molecular diagnosis of genetic disorders.

Retrotransposition disrupting EBP in a girl and her mother with X-linked dominant chondrodysplasia punctata.

X-linked dominant chondrodysplasia punctata (CDPX2) is a rare congenital disorder caused by pathogenic variants in EBP on Xp11.23. We encountered a girl and her mother with CDPX2-compatible phenotypes including punctiform calcification in the neonatal period of the girl, and asymmetric limb shortening and ichthyosis following the Blaschko lines in both subjects. Although Sanger direct sequencing failed to reveal a disease-causing variant in EBP, whole genome sequencing (WGS) followed by Manta analysis identified a ~ 4.5 kb insertion at EBP exon 2 of both subjects. The insertion was associated with the hallmarks of retrotransposition such as an antisense poly(A) tail, a target site duplication, and a consensus endonuclease cleavage site, and the inserted sequence harbored full-length SVA_F1 element with 5'- and 3'-transductions containing the Alu sequence. The results imply the relevance of retrotransposition to the human genetic diseases and the usefulness of WGS in the identification of retrotransposition.

Global developmental delay, systemic dysmorphism and epilepsy in a patient with a de novo U2AF2 variant.

U2 small nuclear RNA auxiliary factor 2 (U2AF2) is an essential pre-mRNA splicing factor in an early step of splicing. Alternative splicing plays an important role in neuronal development, and disorders of RNA processing steps are implicated in neurological disorders. Recently, the large trio whole-exome sequencing study reported U2AF2 as a novel gene significantly associated with developmental disorders: however, the clinical details of patients with U2AF2 variants were not available. Here, we report an individual with a de novo U2AF2 variant (c.445C>T, p.(Arg149Trp)) using trio-based whole-exome sequencing. This residue was positioned in the RNA recognition motif 1 which recognizes a polypyrimidine-tract splice site signal. The patient showed global developmental delay, intellectual disability, epilepsy, short stature, microcephaly, facial dysmorphism, intermittent exotropia, bilateral ptosis, muscle hypotonia and thin corpus callosum, indicating that U2AF2-related disorder could include systemic dysmorphisms, epilepsy and brain malformation along with global developmental delay.

Comprehensive genetic analysis confers high diagnostic yield in 16 Japanese patients with corpus callosum anomalies.

Corpus callosum anomalies (CCA) is a common congenital brain anomaly with various etiologies. Although one of the most important etiologies is genetic factors, the genetic background of CCA is heterogenous and diverse types of variants are likely to be causative. In this study, we analyzed 16 Japanese patients with corpus callosum anomalies to delineate clinical features and the genetic background of CCAs. We observed the common phenotypes accompanied by CCAs: intellectual disability (100%), motor developmental delay (93.8%), seizures (60%), and facial dysmorphisms (50%). Brain magnetic resonance imaging showed colpocephaly (enlarged posterior horn of the lateral ventricles, 84.6%) and enlarged supracerebellar cistern (41.7%). Whole exome sequencing revealed genetic alterations in 9 of the 16 patients (56.3%), including 8 de novo alterations (2 copy number variants and variants in ARID1B, CDK8, HIVEP2, and TCF4) and a recessive variant of TBCK. De novo ARID1B variants were identified in three unrelated individuals, suggesting that ARID1B variants are major genetic causes of CCAs. A de novo TCF4 variant and somatic mosaic deletion at 18q21.31-qter encompassing TCF4 suggest an association of TCF4 abnormalities with CCAs. This study, which analyzes CCA patients usung whole exome sequencing, demonstrates that comprehensive genetic analysis would be useful for investigating various causal variants of CCAs.

Genetic and phenotypic analysis of 101 patients with developmental delay or intellectual disability using whole-exome sequencing.

Whole-exome sequencing (WES) enables identification of pathogenic variants, including copy number variants (CNVs). In this study, we performed WES in 101 Japanese patients with unexplained developmental delay (DD) or intellectual disability (ID) (63 males and 38 females), 98 of them with trio-WES. Pathogenic variants were identified in 54 cases (53.5%), including four cases with pathogenic CNVs. In one case, a pathogenic variant was identified by reanalysis of exome data; and in two cases, two molecular diagnoses were identified. Among 58 pathogenic variants, 49 variants occurred de novo in 48 patients, including two somatic variants. The accompanying autism spectrum disorder and external ear anomalies were associated with detection of pathogenic variants with odds ratios of 11.88 (95% confidence interval [CI] 2.52-56.00) and 3.46 (95% CI 1.23-9.73), respectively. These findings revealed the importance of reanalysis of WES data and detection of CNVs and somatic variants in increasing the diagnostic yield for unexplained DD/ID. In addition, genetic testing is recommended when patients suffer from the autism spectrum disorder or external ear anomalies, which potentially suggests the involvement of genetic factors associated with gene expression regulation.

Identification of a deep intronic POLR3A variant causing inclusion of a pseudoexon derived from an Alu element in Pol III-related leukodystrophy.

Pseudoexon inclusion caused by deep intronic variants is an important genetic cause for various disorders. Here, we present a case of a hypomyelinating leukodystrophy with developmental delay, intellectual disability, autism spectrum disorder, and hypodontia, which are consistent with autosomal recessive POLR3-related leukodystrophy. Whole-exome sequencing identified only a heterozygous missense variant (c.1451G>A) in POLR3A. To explore possible involvement of a deep intronic variant in another allele, we performed whole-genome sequencing of the patient with variant annotation by SpliceAI, a deep-learning-based splicing prediction tool. A deep intronic variant (c.645 + 312C>T) in POLR3A, which was predicted to cause inclusion of a pseudoexon derived from an Alu element, was identified and confirmed by mRNA analysis. These results clearly showed that whole-genome sequencing, in combination with deep-learning-based annotation tools such as SpliceAI, will bring us further benefits in detecting and evaluating possible pathogenic variants in deep intronic regions.

A de novo variant in RAC3 causes severe global developmental delay and a middle interhemispheric variant of holoprosencephaly.

RAC3 is a member of the Rho GTPases family, which has important regulatory functions in aspects of neuronal morphogenesis. Rho GTPases show a conformational change in two regions (switch I and II) through GTP binding, which provides a platform for selective interactions with functionally diverse proteins. Missense variants in the switch I and II regions of RAC3 were recently suggested to cause severe intellectual disability and brain malformations. Here, we report an individual with a novel de novo RAC3 variant (c.101 C>G, p.(Pro34Arg)), which substitutes for an evolutionarily conserved amino acid within the switch I region. The patient showed severe global developmental delay, intellectual disability, epilepsy, and laryngeal dystonia. An imaging study revealed characteristic brain dysplasia, including coexistence of the middle interhemispheric variant of holoprosencephaly and brainstem dysmorphism. Our study supports that RAC3 variants cause syndromic neurodevelopmental disorders and brain structural abnormality, and expands the phenotypic spectrum of RAC3-related disorders.

Identification of de novo CSNK2A1 and CSNK2B variants in cases of global developmental delay with seizures.

Casein kinase 2 (CK2) is a serine threonine kinase ubiquitously expressed in eukaryotic cells and involved in various cellular processes. In recent studies, de novo variants in CSNK2A1 and CSNK2B, which encode the subunits of CK2, have been identified in individuals with intellectual disability syndrome. In this study, we describe four patients with neurodevelopmental disorders possessing de novo variants in CSNK2A1 or CSNK2B. Using whole-exome sequencing, we detected two de novo variants in CSNK2A1 in two unrelated Japanese patients, a novel variant c.571C>T, p.(Arg191*) and a recurrent variant c.593A>G, p.(Lys198Arg), and two novel de novo variants in CSNK2B in Japanese and Malaysian patients, c.494A>G, p.(His165Arg) and c.533_534insGT, p.(Pro179Tyrfs*49), respectively. All four patients showed mild to profound intellectual disabilities, developmental delays, and various types of seizures. This and previous studies have found a total of 20 CSNK2A1 variants in 28 individuals with syndromic intellectual disability. The hotspot variant c.593A>G, p.(Lys198Arg) was found in eight of 28 patients. Meanwhile, only five CSNK2B variants were identified in five individuals with neurodevelopmental disorders. We reviewed the previous literature to verify the phenotypic spectrum of CSNK2A1- and CSNK2B-related syndromes.

De novo variants in SETD1B are associated with intellectual disability, epilepsy and autism.

SETD1B (SET domain containing 1B) is a component of SET1 histone methyltransferase complex, which mediates the methylation of histone H3 on lysine 4 (H3K4). Here, we describe two unrelated individuals with de novo variants in SETD1B identified by trio-based whole exome sequencing: c.5524C>T, p.(Arg1842Trp) and c.5575C>T, p.(Arg1859Cy). The two missense variants occurred at evolutionarily conserved amino acids and are located within the SET domain, which plays a pivotal role in catalyzing histone methylation. Previous studies have suggested that de novo microdeletions in the 12q24.3 region encompassing SETD1B were associated with developmental delays, intellectual disabilities, autism/autistic behavior, large stature and craniofacial anomalies. Comparative mapping of 12q24.3 deletions refined the candidate locus, indicating KDM2B and SETD1B to be the most plausible candidate genes for the pathogenicity of 12q24.3 deletion syndrome. Our cases showed epilepsy, developmental delay, intellectual disabilities, autistic behavior and craniofacial dysmorphic features, which are consistent with those of individuals with de novo 12q24.31 deletions. Therefore, our study suggests that SETD1B aberration is likely to be the core defect in 12q24.3 deletion syndrome.

De novo KIF1A mutations cause intellectual deficit, cerebellar atrophy, lower limb spasticity and visual disturbance.

Recently, de novo KIF1A mutations were identified in patients with intellectual disability, spasticity and cerebellar atrophy and/or optic nerve atrophy. In this study, we analyzed a total of 62 families, including 68 patients with genetically unsolved childhood cerebellar atrophy, by whole-exome sequencing (WES). We identified five de novo missense KIF1A mutations, including only one previously reported mutation (p.Arg316Trp). All the mutations are located in the motor domain of KIF1A. In all patients, initial symptom onset was during the infantile period, and included developmental delay in three patients and gait disturbance in two. Thereafter, they showed gait disturbances, exaggerated deep tendon reflexes, cerebellar symptoms and cerebellar atrophy on brain magnetic resonance imaging. Four patients showed lower limb spasticity, upper limb clumsiness and visual disturbances. Nerve conduction study revealed peripheral neuropathy in three patients. This study further delineates clinical features of de novo KIF1A mutations. Genetic testing of KIF1A should be considered in children with developmental delay, cerebellar atrophy and pyramidal features.

[Recurrent posterior reversible encephalopathy due to vasospasm and cerebral hypoperfusionin in acute leukemia: a case report].

We report the case of a 4-year-old girl who presented with recurrent posterior reversible encephalopathy syndrome (PRES). She was diagnosed with B-precursor acute lymphocytic leukemia (ALL), and was administered remission-induction chemotherapy. On day 28 of the induction therapy, she experienced seizure and prolonged unconsciousness. Blood pressure was slightly elevated. MRI revealed cortical cytotoxic edema in the right temporal and occipital lobes. In the right occipital white matter the lesion with vasogenic edema also existed. Three days later, MRI showed vasogenic edema in subcortical white matter of the right temporal right occipital and bilateral occipital lobes. The lesions had receded with time. Since the seizure occurred, the chemotherapy had been discontinued. The episodes of seizure and prolonged consciousness recurred 22 days later. MRI revealed vasogenic edema in the right occipital lobe, and MR angiography demonstrated vessel irregularity and reduced branch visualization in the middle and posterior cerebral arteries. Arterial spin-labeling (ASL) showed hypoperfusion in both occipital lobes. It suggests that vasoconstriction and hypoperfusion could lead to recurrent PRES in this case. It is possible that ASL might be more sensitive than MRI in detecting the lesions of PRES. It should be noted that PRES might recur in leukemia.

Case Report: Novel compound heterozygous TPRKB variants cause Galloway-Mowat syndrome.

Background: Galloway-Mowat syndrome (GAMOS) is a rare genetic disease characterized by early-onset nephrotic syndrome and microcephaly with central nervous system abnormalities. Pathogenic variants in genes encoding kinase, endopeptidase, and other proteins of small size (KEOPS) complex subunits cause GAMOS. The subunit TPRKB (TP53RK binding protein) has been reported in only two patients with GAMOS with homozygous missense variants. Clinical report: Herein, we described a three-year-old male with GAMOS. He exhibited developmental delay, developmental regression, microcephaly, distinctive facial features, skeletal abnormalities, and epilepsy. Brain magnetic resonance imaging revealed progressive brain atrophy, delayed myelination, T2-hypointense signals in the thalamus, and multiple intracranial abnormal signals on diffusion-weighted imaging. He presented with relapsing nephrotic proteinuria exacerbated by upper respiratory tract infections and progressive renal function decline. Exome sequencing identified compound heterozygous missense and frameshift variants in TPRKB: c.224dup, p.(Ser76IlefsTer3) and c.247C>T, p.(Leu83Phe). Conclusions: Our study supports that pathogenic TPRKB variants cause KEOPS complex-related GAMOS.

A novel de novo TMEM63A variant in a patient with severe hypomyelination and global developmental delay.

BACKGROUND: Heterozygous variants in TMEM63A have been recently identified as the cause of infantile-onset transient hypomyelination. To date, four TMEM63A variants have been reported in five patients. These patients exhibited favorable clinical course, developmental progress, and completion of myelination. CASE REPORT: The patient was a 5-year-old girl with severe global developmental delay, absent speech, no turning over, no gazing, hypotonia, and daily episodes of autonomic seizures. Brain MRI showed hypomyelination of deep and subcortical white matter that appeared hyperintense in T2-weighted imaging from 2 months of age and that showed no change at 4 years of age. Exome sequencing of the patient and her parents revealed a novel de novo missense variant, NM_014698.3:c.1658G>T, p.(Gly553Val), in the TMEM63A gene, which was confirmed by Sanger sequencing. The variant has not been registered in public databases, and it substitutes a highly conserved glycine residue located in a pore-lining transmembrane helix. No other candidate variants were identified. CONCLUSIONS: Although TMEM63A variants are generally thought to cause transient hypomyelination with favorable developmental progress, identification of a de novo TMEM63A variant in our patient suggests that the TMEM63A-related clinical spectrum is broad and includes severe developmental delay with seizures.

Compound heterozygous ADAMTS9 variants in Joubert syndrome-related disorders without renal manifestation.

BACKGROUND: Ciliopathies are the outcomes of defects of primary cilia structures and functions which cause multisystemic developmental disorders, such as polycystic kidney disease, nephronophthisis, retinitis pigmentosa, Joubert syndrome (JS), and JS-related disorders (JSRD) with additional organ involvement including oral-facial-digital syndrome and so on. They often share common and unexpected phenotypic features. CASE PRESENTATION: We report a 4-year-old-boy case with compound heterozygous variants of ADAMTS9. Unlike the cases with ADAMTS9 variants in the previous report, which identified that homozygous variants of ADAMTS9 were responsible for nephronophthisis-related ciliopathies in two cases, the current case did not have nephronophthisis nor renal dysfunction, and his clinical features, such as oculomotor apraxia, hypotonia, developmental delay, bifid tongue, and mild hypoplasia of cerebellar vermis indicated JSRD. CONCLUSIONS: The case suggested a possible association between the clinical presentation of JSRD and ADAMTS9-related disease, and it shows a wide spectrum of ADAMTS9 phenotype.

Familial periodic paralysis associated with a rare KCNJ5 variant that supposed to have incomplete penetrance.

BACKGROUND: The periodic paralyses are a group of skeletal muscle channelopathies caused by variants in several ion channel genes. Potassium Inwardly Rectifying Channel Subfamily J Member 5 (KCNJ5) encodes the G-protein-activated inwardly rectifying potassium channel 4 (Kir3.4) and the heterozygous KCNJ5 variants cause familial hyperaldosteronism and long QT syndrome (LQTS). Recent studies suggested that variants in KCNJ5 are also causative for Andersen-Tawil syndrome, which showed periodic paralysis and characteristic electrocardiogram features. CLINICAL REPORT: We found a heterozygous KCNJ5 variant c.1159G > C, p.(Gly387Arg) in an individual with familial periodic paralysis using exome sequencing. Sanger sequencing revealed that this variant was inherited from his affected mother. The same variant had been previously found in two cases of familial LQTS or Andersen-Tawil syndrome, and functional analysis suggested that this variant might have loss of function effect on channel activity. However, the allele frequency of c.1159G > C variant in an East Asian population of public databases ranged from 0.21% to 0.25%, indicating possible incomplete penetrance. CONCLUSIONS: Our two patients expand the phenotypic spectrum associated with the c.1159G > C KCNJ5 variant, though the variant has very low penetrance.

Novel ALG12 variants and hydronephrosis in siblings with impaired N-glycosylation.

BACKGROUND: ALG12-CDG is a rare autosomal recessive type I congenital disorder of glycosylation (CDG) due to pathogenic variants in ALG12 which encodes the dolichyl-P-mannose:Man-7-GlcNAc-2-PP-dolichyl-alpha-6-mannosyltransferase. Thirteen patients from unrelated 11 families have been reported, most of them result in broad multisystem manifestations with clinical variability. It is important to validate abnormal glycosylation to establish causal relationship. CASE REPORT: Here, we report two siblings with novel compound heterozygous variants in ALG12: c.443T>C, p.(Leu148Pro) and c.412_413insCGT, p.(Gln137_Phe138insSer). Both patients showed global developmental delay, microcephaly, hypotonia, failure to thrive, facial dysmorphism, skeletal malformations and coagulation abnormalities, which are common in ALG12-CDG. In addition, one of our patients showed left hydronephrosis, which is a novel clinical feature in ALG12-CDG. Brain MRI showed hypoplasia of cerebrum, brain stem and cerebellar vermis in both patients. N-glycosylation defects of trypsin digested transferrin peptides were revealed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), and electrospray ionization MS verified the lack of N-glycans in transferrin. CONCLUSIONS: The present study can add hydronephrosis to phenotypic spectrum of ALG12-CDG. Since the symptoms of ALG12-CDG are quite diverse, the combination of whole-exome sequencing and transferrin glycopeptide analysis with MS, can help diagnosis of ALG12-CDG.

A de novo TOP2B variant associated with global developmental delay and autism spectrum disorder.

BACKGROUND: TOP2B encodes type II topoisomerase beta, which controls topological changes during DNA transcription. TOP2B is expressed in the developing nervous system and is involved in brain development and neural differentiation. Recently, a de novo missense TOP2B variant (c.187C>T) has been identified in an individual with neurodevelopmental disorder (NDD). However, the association between TOP2B variants and NDDs remains uncertain. METHODS: Trio-based whole-exome sequencing was performed on a 7-year-old girl, presenting muscle hypotonia, stereotypic hand movements, epilepsy, global developmental delay, and autism spectrum disorder. Brain magnetic resonance images were normal. She was unable to walk independently and spoke no meaningful words. RESULTS: We found a de novo variant in TOP2B (NM_001330700.1:c.187C>T, p.(His63Tyr)), which is identical to the previous case. The clinical features of the two individuals with the c.187C>T variant overlapped. CONCLUSION: Our study supports the finding that TOP2B variants may cause NDDs.