Novel Copy Number Deletion Involving NUS1 Associated With Epilepsy, Tremor, and Intellectual Disability

ABSTRACT

Copy number variations (CNVs) contribute to various disorders including intellectual disability, developmental disorders, and cancer. This study identifies a de novo 2.62 Mb deletion at 6q22.1_q22.31, implicating the NUS1 gene in epilepsy, spinal abnormalities, and intellectual disability, thereby expanding its known phenotypic associations.

1. Introduction

Copy number variations (CNVs) have been linked to a variety of genetic disorders and complex diseases and contribute to phenotypic differences and susceptibility to diseases. For example, certain CNVs are associated with intellectual disabilities, developmental disorders, and cancers. Understanding these associations can help in identifying genetic factors that contribute to these conditions.

The NUS 1 (nuclear undecaprenyl pyrophosphate synthase 1) gene encodes the Nogo‐B receptor (NgBR), a subunit that is essential protein for dolichol synthesis. Dolichol serves as a carrier for the oligosaccharide chain of N‐linked glycosylation, which contributes to proper folding and quality control of proteins [1]. In addition to cellular dolichol synthesis and protein glycosylation, NgBR is responsible for lysosomal cholesterol accumulation. Specifically, lysosomal cholesterol accumulation contributes to movement deficits associated with NUS1 haploinsufficiency [2]. NUS1 was among many candidate genes that were found to be associated with developmental and epileptic encephalopathy (DEE), a group of conditions characterized by co‐occurrence of epilepsy and intellectual disability [3]. A previous study reported six individuals with seizures and overlapping microdeletion on 6q22.1_q22.31, narrowing the critical region to 250 kb. This region encompasses NUS1 and the promoter of SLC35F1, which were important contributors to tremors and epilepsy [4]. Variants of NUS1 have also been linked with ataxia [5]. In some less common cases, NUS1 contributes to parkinsonism, scoliosis, gestational diabetes mellitus, and psychosis [5, 6, 7].

2. Case History and Examination

The subject is a 33‐year‐old man presenting with mild‐to‐moderate intellectual disabilities; the family has spent a lifetime on an odyssey to understand the subject's condition and to collaborate with clinicians to develop the best treatment strategy. The subject has mild facial dysmorphology that includes large ears. He also has excessive drooling. He has scoliosis, gait disturbance, slender build, slender fingers, and a history of extensive tremors. He was born to a non‐consanguineous family. He has a healthy and neurotypical younger male sibling. He presents with global developmental delays, attention‐deficit disorder, hyperactivity, impairment of fine and gross motor coordination, generalized hypotonia, and temper tantrums. Anxiety, impulsivity, and depression were also reported and he was diagnosed with anxiety and mood arousal regulation disorder in 2007. Neuropsychological evaluation was remarkable for poor attention span. Family history is noted for leukemia in his paternal grandfather and possibly autism in his father's sister's son. There is a history of autism spectrum disorder in both the paternal and maternal side of the family.

The proband was born first in birth order from an unremarkable and uncomplicated pregnancy, other than light spotting in the first trimester. He was born at full term via normal, spontaneous vaginal delivery with a birth weight of 3756 g (95th percentile). The perinatal period was uneventful. The proband's first febrile seizure was noted at 11–12 months of age. The seizure was mild and lasted 10 minutes in duration. He then had multiple seizures after 17 months of age. Recurrent seizures consist of staring, and repetitive jerks of the head without dropping, that lasted a few seconds in duration. These episodes were very frequent and may occur 10–20 times over a period of 30 min. Ethosuximide was not effective in treating seizures and was replaced with valproic acid with an improved response noted. He had a history of language disorder speech delay and an attention‐deficit disorder. During an early clinic visit as a child, the boy was observed to be social but nonverbal. He had absent speech but could communicate using simple hand signing. EEG and CT scans were first conducted at 1 year of age and were both unremarkable. Thirty‐two years ago, chromosomal studies were reported as unremarkable, although this would have been karyotype only.

At 3 years, 3 months of age, referral to medical genetics was made. The proband's height, weight, and head circumference were at the 85th, 75th, and 45th percentile, respectively. The neurological exam showed hyperactivity, impairment of fine and gross motor coordination, and generalized hypotonia. Neuropsychological evaluation was remarkable for poor attention span and learning and intellectual disability. His expressive language was assessed as being less than an 8‐month level and his receptive language was at 2–2 ½ year level. A Test of Nonverbal Intelligence Test (TONI‐4) was administered twice to assess abstract reasoning and problem solving, of which he scored 85 and 119, respectively on an index score of 100 and a standard deviation of 15. At the same time, cytogenetic evaluation was performed to rule out Angelman syndrome, (AS). Analyses indicated that the critical DNA region for AS had not been deleted; although it was noted that additional DNA markers were needed to completely rule out AS.

At 4 years of age, he had a weight, height, and head circumference of 50th, 90th, and 45th percentile, respectively. His weight percentile dropped from 85th to 50th, and to this day he is lean. He did not have expressive language and could only say “hi” or “bye.” The subject was able to follow one and two‐step commands and had poor coordination. The proband was referred to a psycholinguistic evaluation and was diagnosed with seizure disorder and developmental receptive and expressive language disorder. At 6 years of age, EEG was indicative of diffuse cerebral dysfunction, and consistent with a diagnosis of primary generalized epilepsy. He only developed limited spoken language despite receiving nonverbal cognitive scores in the average range of his age. Valproic acid was increased to 500 mg bid, from 125 mg bid and carnitine was recommended. By 10 years of age, the proband's nonverbal mental age did not continue to maintain the same rate of change as his physical age, dropping to an IQ of 68, as measured by the Leiter‐R. He also underwent back surgery that year for correction of severe kyphosis.

The next follow‐up psychiatric visit was when the subject was 17 years of age. He was noted to be ambulatory with abnormal gait, dysarthric, with a continuance of tremors and seizures. Due to his significant hand tremors, he required assistance with his daily living routine. He did not exhibit self‐injurious behavior and psychosis was not observed.

The proband was seen again for neuropsychological evaluation at 21 years of age. His general intelligence function was assessed with the Stanford Binet Intelligence Scales, Fifth Edition. He obtained a verbal IQ of 43 (< 1st percentile), a nonverbal IQ of 52 (< 1st percentile), and a combined IQ of 45 (< 1st percentile). This score placed him in the moderately delayed range of general intelligence function. His adaptive behavior was also estimated with the Vineland Adaptive Behavioral Scales, Second Edition, which placed him at an approximate age equivalent of 3 years 7 months. His adaptive skills, including communication, daily living skills, and socialization, were hindered in part by delays in communication skills and significant tremors.

In 2023, and at 31 years of age, he returned again to the George A. Jervis Clinic at the New York Institute for Basic Research, Staten Island, New York for an updated psychological and genetic evaluation. The proband continued to have extensive tremors which affected his daily functioning such as feeding himself. He also had choreoathetosis and possible dyskinesias. Ataxia was not noted.

3. Methods

Our differential diagnoses include primary generalized epilepsy, Fragile X syndrome, and Angelman syndrome. Fragile X testing was conducted and reported as negative. At the same time, cytogenetic evaluation was performed to rule out Angelman syndrome (AS). Analyses indicated that the critical DNA region for AS had not been deleted. EEG was indicative of diffuse cerebral dysfunction and consistent with a diagnosis of primary generalized epilepsy. A whole‐genome single‐nucleotide polymorphism (SNP) microarray was performed on the subject and both parents as a trio. An interstitial deletion on chromosome 6 was found on the proband, while both maternal and paternal microarray revealed a normal copy number. This analysis was used to determine the gene anomaly contributing to the phenotype. Brain MRI was unattainable because of the steel rods placed following kyphosis correction surgery. The proband's lipid panel was obtained (Table 1) and showed abnormally elevated levels of total cholesterol, triglycerides, LDL cholesterol, and low HDL cholesterol, but a normal BMI of 21.7 kg/m². Additional lab testing did not find any abnormality with his oxysterol profile (see Table 2). Table S1 shows a comprehensive list of the current medications of the proband. Clonazepam is taken to control epilepsy, zonisamide for tremors, memantine for ataxia, and quetiapine to manage behaviors.

TABLE 1.

Lipid panel.

	Value (high/low)	Reference range
Cholesterol, total	252 (H)	< 200 mg/dL
HDL cholesterol	35 (L)	> OR = 40 mg/dL
Triglycerides	272 (H)	< 150 mg/dL
LDL cholesterol	172 (H)	< 100 mg/dL
CHOL/HDLC ratio	7.2 (H)	< 5.0
Non‐HDL cholesterol	217 (H)	< 130 mg/dL

TABLE 2.

Oxysterols.

Oxysterols	Concentration (nmol/m)	Reference value (nmol/m)
Cholestane‐3β,5ɑ,6β‐triol	< 0.055	≤ 0.070
7‐Ketocholesterol	< 0.038	≤ 0.100
Lyso‐sphingomyelin	< 0.006	≤ 0.100

4. Conclusion and Results

A whole‐genome SNP microarray was performed as a trio on the subject, mother, and father. A de novo 2.62 Mb interstitial deletion of 6q22.l_q22.31 was detected, which is interpreted as pathogenic, as seen in Table 3. This interval includes nine OMIM genes (VGLL2, ROSl, GOPC, NUS1, CEP85L, PLN, MCM9, ASFlA, and MANlAl) summarized in Table 4 [8, 9, 10, 11, 12, 13, 14, 15, 16, 17]. In addition, the microarray also identified a 1.16 mb interstitial duplication of 7p21.2_ p21.1 and 77 kb interstitial duplication of 22q12.1_q12.1 in the proband. This interval includes six OMIM genes (BZW2, TSPAN13, AGR2, AGR3, AHR, and SNX13). At this time, no clinically established disorders have been reported with duplication of this region.

TABLE 3.

NUS1 variant finding.

Gene	Chr	Position	HGVS DNA reference	HGVS protein reference	Variant type	Predicted effect	dbSNP/dbVar ID	Genotype
NUS1	6	6q22.31	NM_138459.5:g.117344163_119962769del	NP_612468.1	Deletion	Haploinsufficiency	n/a	Heterozygous

TABLE 4.

The genes included in the de novo 2.62 Mb pathogenic interstitial deletion of 6q22.l_q22.31.

Genes	Description	Conditions	Pathogenicity
VGLL2	Vestigial like family member 2	Spindle cell rhabdomyosarcoma; pleomorphic rhabdomyosarcoma	Unknown
ROS1	ROS proto‐oncogene 1, receptor tyrosine kinase	Susceptibility to lung cancer	Unknown
GOPC	Golgi‐associated PDZ And coiled‐coil motif containing	Spermatogenic failure 9 and acute laryngitis	Unknown
NUS1	NUS1 dehydrodolichyl diphosphate synthase subunit	Congenital disorder of glycosylation, Type Iaa, and autosomal dominant type 55 intellectual disability with seizures	Likely pathogenic
CEP85L	Centrosome protein 85 like	Lissencephaly 10 and lissencephaly	Likely pathogenic
PLN	Phospholamban	Cardiomyopathy, dilated, 1P and cardiomyopathy, familial hypertrophic, 18	Likely pathogenic
MCM9	Minichromosome maintenance 9 homologous recombination repair factor	Ovarian dysgenesis 4 and premature ovarian failure 1	Likely pathogenic
ASF1A	Anti‐silencing function 1A histone chaperone	Cataract 34, multiple types and alpha‐thalassemia	Unknown
MAN1A1	Mannosidase alpha class 1A member 1	Congenital disorder of glycosylation, type IIu	Unknown

Currently, the management plan for the subject focuses on symptomatic treatment and supportive care. The subject attends physical therapy twice a week. The patient is periodically assessed to monitor symptom progression.

5. Discussion

NUS1 is classically associated with congenital disorder of glycosylation, Type Iaa, and autosomal dominant type 55 intellectual disability with seizures [2, 3]. So far, there have been reports of nine de novo NUS1 variants linked to developmental and epileptic encephalopathy [5]. Individuals with congenital disorders of glycosylation are susceptible to a variety of different symptoms across several different organ systems, which include developmental delays, poor growth, nerve damage, endocrine dysfunction, and facial dysmorphism. There are over 130 different diseases linked to dysfunctions in the glycosylation pathway, many of which do not have any treatment [18]. Although some congenital disorders of glycosylation can be found to have elevated oxysterols [19], this did not appear to be the case in our patient, perhaps suggesting that the intact copy of NUS1 on the other chromosome is sufficient to prevent substantial oxysterol accumulation.

ASF1A is a gene responsible for encoding a histone chaperone protein belonging to the H3/H4 family and regulates nucleosome assembly [20]. All three studies reported in Clinvar that involve ASF1A deletion have a variant length over 1 kb involving multiple genes, making it difficult to attribute the connection between ASF1A to the neurodevelopmental symptoms. VGLL2 is a gene that encodes for the transcription factor of vestigial‐like protein 2 that is associated with skeletal muscle development [4]. Variants including deletions in this gene had other genes deleted including NUS1 [21]. Thus, the precise contribution of this gene or its lack thereof could not be determined. Similarly, GOPC is a gene that encodes a Golgi protein that could contribute to infertility [22]. Variants in ClinVar included NUS1 and other genes, making it difficult to determine its contribution. Finally, ROS1, a proto‐oncogene that encodes for either a protein or differentiation factor could contribute to tumor development [23]. Additional variants with ROS1 deletion included NUS1 and other genes and did show phenotypes of delayed speech and language development as well as intellectual disability. Thus, based on our search, the patient's phenotype of seizures, tremors, intellectual disability, and delayed speech and language development seem to be more associated with a NUS1 mutation as opposed to the other OMIM genes.

We report a novel de novo 2.62 Mb interstitial deletion at 6q22.1_q22.31 that implicates the NUS1 gene as a significant contributor to the observed phenotypes. NUS1 classically has been associated with congenital disorders of glycosylation and intellectual disability. However, our findings regarding this deletion suggest a broader phenotypic spectrum for NUS1 variants, encompassing manifestations like kyphosis, choreoathetosis, and possible dyskinesias, which were not notably linked to this gene before. This paints a clearer picture of NUS1's clinical relevance beyond previously known links. The results of this report also add to the building body of evidence that highlights the significance of CNVs in neurological and developmental disorders. Our findings reinforce the importance of genetic analysis in patients presenting with atypical symptoms.

Author Contributions

Jing Y. Hsu: data curation, formal analysis, investigation, writing – original draft. Daniah H. Ibrahim: data curation, formal analysis, investigation, writing – original draft. Riza Ali: data curation, formal analysis, investigation, writing – original draft. Elaine Marchi: data curation. Maureen Gavin: data curation, investigation. Karen Amble: investigation, project administration. Gholson J. Lyon: conceptualization, data curation, funding acquisition, investigation, supervision, writing – review and editing.

Ethics Statement

Subject consent was obtained for research and publication, with approval of protocol #7659 for the Principal Investigator: G.J.L., of the George A. Jervis Clinic by the New York State Psychiatric Institute—Columbia University Department of Psychiatry Institutional Review Board.

Consent

Written patient consent was obtained for research and publication.

Supporting information

Data S1.

Data Availability Statement

The authors confirm that the data supporting the findings of this study are available within the article and its Supporting Information.