Novel de Novo Nonsense Variants in and : Insights into Global Developmental Delay and Autism Spectrum Disorders through Whole Genome Analysis

Introduction

Neurodevelopmental disorders (NDD) are complex disorders with a broad spectrum of clinical and genetic heterogeneity. NDD may present with global developmental delay (GDD), intellectual disability, autism spectrum disorders, communication and learning disorders, motor developmental disorders, epilepsy, sleep disorders, and behavioral disorders. NDD affects 1–3% of the population [1], while GDD is a common clinical finding with an estimated prevalence of 3% among preschool children [2]. Without large-scale advanced genome analysis methods, the underlying etiology of these conditions often remains unknown [3].

De novo causative variants have been reported in an estimated 1% of NDD patients [4,5], and an estimated 1 in 200–400 children with DD are reportedly positive for de novo causative variants (DD) [6]. This number is expected to increase as more causative variants are discovered [3,7]. Next-generation sequencing is an effective first-tier test in patients with DD [3,8]. Trio joint WGS is particularly effective in the evaluation of NDD, as it enables comprehensive genomic and phasing analysis. Herein, we present the phenotypic spectrum and joint WGS analysis of a patient with GDD, autistic features, and hypotonia.

Case Report

A 6-year-old girl and her unaffected mother and father (Figure 1) consented to participate in the CROseq genome program at the Department of Pediatrics, University Hospital Centre Zagreb. The CROseq Genome Program is a collaborative research program between Brigham and Women’s Hospital (BWH) (Boston, USA) and the Department of Pediatrics, University Hospital Centre Zagreb (Zagreb, Croatia), supported by the Mila Za Sve Foundation (Rijeka, Croatia). Participants were enrolled in the study at the University Hospital Center Zagreb.

The proband presented with GDD, hypotonia, and autistic features. She was the second child from healthy and non-consanguineous parents with South European ancestry. The family history was negative for GDD, hypotonia, and autistic features. She was conceived by in vitro fertilization and born after an uneventful pregnancy, except for weaker fetal movements in comparison to the mother’s previous pregnancy. At 2 months old, the patient presented generalized hypotonia and poor head control. Despite her muscle hypotonia, her deep tendon reflexes were normal. She also had right hip dysplasia and left hip subluxation. During infancy, she had feeding difficulties, failure to thrive, absence of a social smile, and poor eye contact, fixation, and tracking of faces and objects.

At 6 and 8 months she could hold her head up and roll her head, respectively. She could independently sit at 15 months and walk at 30 months. She had delayed visual maturation and alternating exotropia. At 2 years of age, a visual evoked potential study revealed cortical visual impairment. At 3 years old, a brain MRI scan showed a moderate reduction in periventricular white matter, which was more prominent in the frontal lobe (Figure 2). Subsequently, the frontal horns of the lateral ventricles were enlarged. The brain MRI also displayed posterior corpus callosum and bilateral optic nerve hypoplasia. Chiari malformation type I with normal myelinization was also notable in MRI. Later, electroencephalography (Nihon Kohden, Neurofax EEG-1200K; Zagreb, Croatia) was performed to assess the development of brain activity and monitor possible changes. EEG signals were recorded at a sampling rate of 200–10 000 Hz. The patient’s forehead was cleaned with alcohol-soaked cotton to keep the impedance of each channel electrode at less than 5 kΩ. The electrodes were placed according to FPz-F9 and FPz-AF7 of the international 10–20 system, encompassing the brain in the same hemisphere. EEG had showed epileptiform activity (Figure 3). However, there was no seizure history. Metabolic workup was within normal limits.

At 7 years of age, the patient’s weight and height measured at 15 kg (Weight SDS −3.49; Weight for Height SDS −2.25; BMI SDS 12.86, standard deviations according to Centers for Disease Control and Prevention) and 108 cm (Height SDS (CDC) −2.67;). Dysmorphic features were present: high forehead/frontal bossing, broad nasal bridge, epicanthic folds, low-set ears, macro-tia, overfolded helix, short nose, and thin upper lip vermilion (Figure 4). She had enlarged frontal tubers, epicanthus, and upper lip frenulum (Figure 4). She also exhibited moderate neuro-developmental delay, including cognitive impairment and learning difficulties. Although she could understand simple, familiar, and situationally supported commands, she had not developed speech or verbal communication by the age of 7 years. She could only communicate by smiling, crying, and gesturing. Her eye contact was poor and short. Visual attention was also easily distracted; however, she was particularly interested in objects that produce sound. She had visual-spatial perception difficulties. She was unable to climb stairs independently and often crawled upstairs. Her fine motor skills were poor, she required feeding assistance, and she did not have control over her sphincter muscles. She had sleep disturbances, including early-morning awakening with difficulty in falling back to sleep.

The karyotype analysis and CMA were normal. Therefore, she was enrolled in the CROseq genome program. The trio-WGS joint analysis was performed at BWH based on the phenotypical features of the proband. The following Human Phenotype Ontology (HPO) terms were used for analysis: generalized hypotonia (HP: 0001290), motor delay (HP: 0001270), delayed speech and language development (HP: 0000750), cognitive impairment (HP: 0100543), global developmental delay (HP: 0001263), autistic behavior (HP: 0000729), EEG with focal epileptiform discharges (HP: 0011185), Arnold-Chiari type I malformation (HP: 0007099), cerebral white matter atrophy (HP: 0012762), and ventriculomegaly (HP: 0002119).

There were no disease-causing variants in the OMIM genes which were associated with these phenotypical findings. However, 2 de novo nonsense heterozygous variants were identified, and Sanger confirmed (Tables 1, 2; Figure 5). The variant AGO3 (NM_024852.4): c.1324C>T (p.Gln442*) is in exon 11, and KHSRP (NM_001366299.1): c.1573C>T (p.Gln525*) is located at exon 15. The gene constraint metrics for AGO3 and KHSRP are pLI=1, o/e = 0.15, and pLI=1, o/e=0.17, respectively; therefore, both genes are predicted to be loss of function intolerant. A knock-out mouse study showed that loss of function in the KHSRP gene leads to impairment in neuronal development [9]. Therefore, loss of function may be a mechanism of disease for KHSRP in the context of the GDD and autistic features. It is not clear whether the AGO3 variant acts in the loss of function manner for the GDD phenotype. The variants were absent in the gnomAD database. They have not been reported in the ClinVar database or any published literature. Because it is not clear whether loss of function serves as the mechanism of disease associated with these genes, the identified variants are considered as presumably deleterious.

Discussion

Joint WGS assessment is a valuable tool for investigation of complex disorders by allowing comprehensive analysis of whole-genomic data and simultaneous phasing investigation of the genome. In this study, trio-WGS joint analysis identified two de novo nonsense variants in a patient with GDD and hypotonia.

The NM_024852.4: c.1324C>T variant is in the AGO3 gene, located on chromosome 1p34.3, and is involved in miRNA processing [10,11], and negative mRNA regulation via a de-capping mechanism [12]. The encoded protein is part of an RNA-induced silencing complex (RISC) but appears to have no function in mRNA cleavage [10,11]. The AGO3 gene is currently not known to be associated with a syndrome in OMIM. However, other members of the Argonaute protein family, AGO1 and AGO2, are OMIM genes with phenotypes similar to those in the proband in our study. AGO1 gene is associated with an autosomal dominant condition called neurodevelopmental disorder with language delay and behavioral abnormalities, with or without seizures (NEDLBAS, MIM # 620292) [13,14]]. It is typically associated with hypotonia, feeding difficulties, and subtle facial dysmorphism. The AGO2 is associated with autosomal dominant Lessel-Kreienkamp syndrome (LESKRES, MIM#619149) [15], characterized by an NDD, GDD, cognitive deficits, speech and language delay, behavioral problems, hypotonia, gait abnormalities, visual defects, and nonspecific dysmorphic facial features. Both AGO1- and AGO2- associated phenotypes share similarities with our proband, particularly AGO2, which includes visual defects, suggesting possible overlapping features with AGO3. We did not observe any phenotype in our AGO3-positive patient that was not present on reported AGO1 and AGO2-associated conditions. A clinical case series described 5 patients with GDD and the potential role of AGO3 in the neurodevelopmental findings [16]. The patients in that study had a deletion of 1p34.3 region encompassing contiguous AGO1 and AGO3 genes. The authors hypothesized that haploinsufficiency of AGO1 and AGO3 genes accounted for the neurocognitive phenotypes, including motor and verbal delay, hypotonia, and mild-to-moderate intellectual disability, as well as dysmorphic features [16]. These phenotypic features in that case series were also present in our proband in this study. Furthermore, 2 patients from this case series had cortical brain atrophy, similar to our patient. Joint laxity was another common feature, as our patient had hip dislocation. These observations suggest a potential role of AGO3 in these common phenotypes. More case collection and experimental studies are required to elucidate the role of AGO3 in disease.

The KHSRP: c.1573C>T is in chromosome 19p13.3. The KHSRP gene encodes an RNA-binding protein that promotes the degradation of a subset of mRNAs [17]. This gene binds the G-rich terminal of the precursor miRNA, which leads to increased precursor miRNA cleavage for maturation [18]. The KHSRP gene is not yet known to be associated with a Mendelian human disease. Experimental studies have shown that KHSRP is expressed in neural tissues, colocalizes with the Dicer complex in axon endings, and impedes axon growth [19]. Loss of KHSRP function in mouse knock-out models can increase axon and dendrite growth [9]. Additionally, KHSRP reportedly regulates genes involved in autism spectrum disorders, such as AUTS2 and GRIN2A [20], which also exhibit increased dendrite density [21]. Based on the findings of these studies on KHSRP, it could be postulated that KHSRP haploinsufficiency may play a role in the autistic features observed in the patient in our study. Comprehensive functional studies are needed to investigate the precise role of KHSRP in disease.

Several genes and molecular pathways are involved in the pathogenesis of GDD and autism [22,23]. Both KHSRP and AGO3 play a role in miRNA metabolism by interacting with DROSHA [18,24,25]. However, their role in GDD etiology, either separately or possibly together, is not known. Experimental studies are needed to investigate the potential role of these genes in neuronal development and disease pathology.

Conclusions

In summary, a comprehensive trio-WGS analysis of a patient with GDD, hypotonia, and autistic features identified a novel and de novo likely pathogenic nonsense variant in each AGO3 and KHSRP. The exact roles of these genes and variants in disease, both separately and possibly together, are not known. It is also not clear whether other genetics and non-genetic modifiers influence the phenotype. Further experimental studies and larger cohorts with detailed clinical descriptions are needed to delineate the functional role of these variants. Sharing reports of these rare conditions is important to communicate these genomic findings in the literature with a larger community and potentially identify similar cases.