Genomic exploration of pediatric neurological disorders: a case series

Abstract

Background

Pediatric neurological disorders include neurodegenerative diseases causing cognitive impairment and vision loss. They are one of the important causes of morbidity and mortality in children with diverse etiologies. Diagnosis is difficult despite genetic work, and a final diagnosis can be achieved in only 60% of cases.

Case presentation

We explore three Indian cases of pediatric neurological diseases (with age presented at the clinic), viz. arthrogryposis (8 years), autism (18 months), and congenital bilateral cataract (3 years), by analyzing clinical exomes. In this work, we attempt to understand rare neurological disorders in an Indian pediatric cohort using exome studies.

Conclusion

We used our benchmarked CONVEX pipeline for screening consensus variants, wherein EIF2B2 was found to be inherently pathogenic. We map the association of variants and genes and disease correlation to neuroleptic malignant syndrome, which matches the phenotype to the cases.

Introduction

Neurological disorders are quite common, with congenital/inborn disorders very difficult to diagnose or detect [1]. Intellectual disability (ID), autism, and other associated disorders are known to affect 1–3% of the world’s population, with an incidence of 1 in 12,000 live births [2]. Although the NCBI's dbSNP (https://www.ncbi.nlm.nih.gov/snp/ last accessed on 1 July 2024) is validated, and ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/ last accessed on 1 July 2024) has a compendium of bona fide records, not all data are reviewed thoroughly. For example, several disease-causing genes that are known, with specific genetic diagnosis, remain elusive in many cases [3], and as these are all heterogeneous groups of disorders, various mutations associated with developmental defects and dysfunction are not properly reviewed and annotated. The last decade has led to a better understanding of disease. Diagnosis is owing to chromosomal breakpoint mapping, multiomics integration, systems genomics approaches, whole-genome array-based copy-number analysis, and nanostring panels, besides the emphasis on understanding the pedigree structure for molecular diagnosis [4]. In this work, we attempt to identify pathogenic mutations in three neurological diseases, viz. arthrogryposis, bilateral congenital cataract with global developmental delay (GDD), and autism spectrum disorders (ASD). The autism spectrum is a neurodevelopmental condition that is visible at the beginning of early childhood and persists throughout life [5]. As it affects the nervous system, the affected person lacks cognitive, emotional, social, and physical health. The severity and duration of symptoms might vary substantially, with severe communication and social interaction issues with repetitive behavior patterns. Knowing the genetic origin of ASD could be one of the most critical aspects of future diagnosis and therapy [6]. Likewise, arthrogryposis is a condition that causes a variety of joint contractures. It is a complicated etiological illness that affects one in 3000 live babies, although the prenatal frequency is higher, signifying a high intrauterine mortality rate [7]. The disease’s genetic diversity has been demonstrated by linking it to 400 distinct genes. Intrinsic/primary/fetal etiology is caused by abnormalities in different body sections, including the brain, nerve cells, muscles, bones, tendons, joints, etc. Among the 400 genes implicated, 9 newly found genes, including CNTNAP1, MAGEL2, ADGRG6, ASXL3, and STAC3, harbor pathogenic variants [8].

Case presentation

Chief complaints

Case 1: a male Indian child with arthrogryposis congenita with delayed development presented to the clinic at 8 years of age.

Case 2: a Male Indian child with microcephaly and autism presented to the clinic at 18 months of age.

Case 3: an male Indian child with bilateral cataract, failure to thrive (FTT), and some developmental delay presented to the clinic at 3.5 years of age.

History of past illness

Case 1: On antenatal ultrasonography, the baby was found to have a short femur and humerus, and the mother had hyperemesis in all trimesters. He was born full term via normal vaginal delivery and had meconium-stained liquor along with crossed leg and contracture of the elbow and bilateral right hamstring and iliotibial band. During 6 days of hospitalization, no hypoglycemia or convulsions were noted. For contracture, he was operated on at the age of 3 years. At 8 years old, he was not able to walk or sit by himself but can do this with support. He received physiotherapy and followed up with an orthopedician but without much benefit.

Case 2: The parents noticed developmental delay at 18 months of age. At present, he is able to walk and run but cannot communicate in sentences, being able to speak only words. He is under follow-up and taking treatment from speech and behavioral therapists but without much improvement.

Case 3: The mother had a fever for 2–3 days without any rash in the first 2 months of pregnancy. While antenatal ultrasound showed signs of early-onset intrauterine growth retardation, he was born preterm with low birth weight (1.7 kg) and admitted into the neonatal intensive care unit (NICU) for 8 days. He has not had regular follow-up with a pediatrician or physiotherapist. Bilateral extraction of cataract was done at 1 year of age.

Personal and family history

Case 1: Our history-taking revealed family history. His sister had a similar disease, but she was only mildly affected compared with him.

Case 2: He was born from a nonconsanguineous marriage without significant family history.

Case 3: He was born from a nonconsanguineous marriage without significant family history.

Physical examination

Case 1: He had GDD and microcephaly (head circumference 50 cm), and other examination findings indicated a broad nose, long toes, laxity of fingers, with hyperextensibility of the knees and elbow.

Case 2: The clinical examination showed microcephaly, hypertonia, failure to recognize things, and significant speech delay.

Case 3: The clinical examination showed microcephaly, FTT, and a small anterior fontanelle with a prominent metopic suture. He had normal motor development but significant speech delay (only cooing).

Laboratory investigations

Case 1: On referral from the previous investigation by a neurologist, an investigation showed normal karyotype, electromyography, and nerve conduction study.

Case 2: Thyroid and routine blood investigations were normal.

Case 3: The ophthalmic examination showed bilateral cataracts (right > left) with intraocular calcification. Other investigations showed normal to complete hemogram, toxoplasma, and other, rubella, cytomegalovirus, and herpes simplex negative, with normal renal and liver function tests except increased alkaline phosphatase level (567 U/L).

Imaging examinations

Case 1: Magnetic resonance imaging (MRI) findings of the brain showed paucity of white matter.

Case 2: MRI findings of the brain were normal.

Case 3: Computed tomography of the brain showed generalized cerebral shrinkage with prominence of cortical sulci and cisternal spaces with bilateral periventricular volume loss and ex vacuo prominence of frontal horns.

Final diagnosis

Case 1

The patient was thought to be suffering from arthrogryposis congenita, most probably due to neurological causes.

Case 2

The diagnosis was a complex autism phenotype with microcephaly, probably due to genetic cause.

Case 3

The diagnosis was GDD with bilateral cataracts to rule out genetic or metabolic etiology.

Treatment

All of them were advised regarding physiotherapy and developmental and stimulation therapy, but none of them were on regular treatment.

Outcome and follow-up

Case 1

The patient was able to walk with difficulty, showing no further deterioration or improvement from the previous level.

Case 2

The patient had some improvement in speech and social communication but still significant delay.

Case 3

The patient had more neurological deterioration, not being able to sit, stand, or communicate.

Discussion

Three variants in SMPD4 are known to be associated with arthrogryposis

SMPD4 is the only known gene implicated in arthrogryposis from our exome sequencing analyses, and furthermore, we found three variants derived from the same gene, SMPD4 (Table 1) [9]. These are found on chromosome 2 (rs766318490, rs780446128, and rs1391542283) with minor allele frequency (MAF) for the first two variants amounting to ≤0.0001%, showing that they are extremely rare variants. The MAF of rs766318490 and rs780446128 in the GnomAD showed minor allele of T = 0.000016/4 and A = 0.000004/1, respectively, while the Allele Frequency Aggregator (ALFA) database showed T = 0.000051/1 and A = 0./0. For rs1391542283, GeneMANIA [10] yielded distinct interactions for SMPD4, and many pathways, associated with transcription regulation, factors, cell adhesion, chromatin binding, and neurodegeneration (Fig. 1). SMPD4 is associated with ceramide and is produced by sphingomyelinases as a secondary messenger in intracellular signaling pathways involved in the cell cycle, differentiation, or death. SMPD4 mediates tumor necrosis factor-stimulated oxidant generation in skeletal muscle showing biallelic loss-of-function mutations in SMPD4, which codes for the neutral sphingomyelinase-3/SMPD4. However, we could not find any SMPD4 variants attributing to pathogenesis from our CONVEX pipeline. Proteomics research on human Myc-tagged SMPD4 overexpression demonstrated localization to both the outer nuclear envelope and the endoplasmic reticulum (ER), and interactions with multiple nuclear pore complex proteins. Fibroblasts from afflicted people had aberrant ER cisternae, suggesting enhanced autophagy, and were more vulnerable to apoptosis under stress circumstances, whereas SMPD4 therapy slowed cell cycle progression. It has been demonstrated that SMPD4 connects membrane sphingolipid homeostasis to cell fate by regulating the crosstalk between the ER and the outer nuclear envelope and that its absence indicates a pathogenic mechanism in microcephaly [11]. The three variants are unreported in non-Asian databases, as they are very rare, highlighting that there have been limited examinations of these variants.

Table 1 List of identified pathogenic variants

Fig. 1

Gene networks and validation of single-nucleotide polymorphisms. A GeneMANIA interaction network (all interactions) with physical interaction network as indicated by pink edges; B phenolyzer map of all genes associated with distinct pathways. The colored genes match epilepsy. The most disease-relevant genes are shown as seed genes alongside predicted genes in the deletion regions. Green lines show that the two node genes belong to the same gene family, whereas yellow lines indicate that they belong to the same biosystem. This pink node represents the diseased phenotypes that we enter regarding our disease. It includes autism spectrum disorders, epilepsy, neuroleptic malignant syndrome, and neuro abnormalities; C Sanger validation results

Pathogenic variants associated with microcephaly and autism

The pathogenic variants were screened for case 2, and a final list of 15 variants was filtered across subpopulation databases and specific phenotype matches based on their MAF and clinical significance. The four pathogenic variants, viz. NM_001104.4(ACTN3):c.1729C > T(p.Arg577Ter); NM_015346.4(ZFYVE26):c.-70A > T; NM_000796.6(DRD3):c.1077C > T(p.His359 =); NM_001369.3(DNAH5):c.2253C > A (p.Asn751Lys), were found at chromosome position 11, 14, 3, and 5, respectively, and were linked with neurodevelopmental disorders such as schizophrenia and structural brain anomalies. A few extremely rare variants with MAF ≤ 0.001 were identified for the filtered set matching the index case. These variants were further searched in the Indian genome variant database, IndiGen (https://clingen.igib.res.in/indigen, last accessed 1 July 2024), in addition to ALFA and GnomAD_exomes reporting these variants (Table 1). However, from the latest ClinVar mapping, we found them to be benign. Further shortlisting to two pathogenic variants (ACTN3) was done and probed to check whether they are associated with inherent pathways. The genes harboring mutations were alpha-actinin-3 (ACTN3), dopamine receptor D3 (DRD3), dynein axonemal heavy chain 5 (DNAH5), and zinc finger five types containing 26 (ZFYVE26), as candidates inherent to congenital bilateral cataract, besides D3 subtype receptor proteins inhibiting adenylyl cyclase pathways (Fig. 1). The receptor is localized to the limbic areas of the brain and associated with cognitive, emotional, and endocrine functions. Several literature studies show that this gene has some association with ASD [12, 13]. A single-nucleotide polymorphism of the DRD3 gene (rs167771) was recently associated with ASD, with different polymorphisms corresponding to varying degrees of behavior [14]. In contrast, the other two genes are normal genes unrelated to neurological disease. We further sought to check whether or not Phenolyzer pathways revealed the diagnosis of PWS and how four genes, viz. DRD3, DNAH5, ZFYVE26, and ACTN3, are associated with the phenotypes represented by human phenotype ontology terms [15]. The branched-chain ketoacid dehydrogenase kinase (BCKDK) is a seed gene on chromosome 16 associated with the mitochondrial protein kinases family and regulates the catabolic pathways for valine, leucine, and isoleucine. By searching for two related types of syndromic ASD, one caused by mutations in BCKDK and the other by mutations in BCKDK, lower BCAA levels may also be detrimental to brain development, as evidenced by the discovery of BCKDH mutations in families with ASD, ID, and seizures. Other genes ACAT1, SLC2AL, ALDH7A1, SCN2A, PSAT1, SCNIA, and KCNJ11 are all connected and may likely cause some neurological disorders. ASD is a complex neurodevelopmental condition characterized by social communication deficits and repetitive behaviors [11, 12]. Recent research has highlighted the involvement of molecular factors in ASD, with particular attention to the glycoprotein Reelin. Encoded by the RELN gene, Reelin plays a crucial role in neuronal migration and synaptic plasticity during brain development, notably in the cerebral cortex and cerebellum, influencing neural circuit formation. Altered Reelin expression in individuals with autism suggests its potential contribution to atypical neural connectivity. The downstream effects of Reelin on molecular pathways, including the modulation of GABAergic interneurons, further enhance its role in autism pathogenesis. Recent findings underscore the importance of considering Reelin’s role in the biological etiology of autism, emphasizing the intricate interplay of genetic and environmental factors [16]. These insights not only advance our understanding of autism’s biological underpinnings but also offer promising avenues for targeted therapeutic interventions, shaping the future of diagnostic and therapeutic strategies for individuals with autism.

Conclusion

Genetic variation attributing to pathogenesis is a significant bottleneck. In this work, we attempted to understand rare neurological disorders in an Indian pediatric cohort using exome studies. While we found that EIF2B2 is inherently pathogenic, we find that neuroleptic malignant syndrome may cause brain damage, which is a matching phenotype. Our study, however, has certain limitations, viz. (1) Parental genotyping or family exome analyses were not done, which could bring candidate germline mutations associated with these disorders. Despite the lack of parental data and the potential for misdiagnosis, this study identifies the inherent pathogenicity of EIF2B2 and its association with neuroleptic malignant syndrome and brain damage. Nonetheless, we acknowledge that our findings are specific to a particular region in India and may not be attributed to the entire population. (2) The potential presence of intronic variants and the limited scope of exome sequencing necessitate further investigation with whole-genome sequencing and functional studies. Identifying EIF2B2 as a disease-causing gene offers a promising avenue for future research and development of targeted therapies for these rare neurological disorders. Small sample sizes may not be typical for the general population, especially in rare diseases. We argue that, if the research population is not diverse, the results may not be applied to other ethnic or demographic groups. The clinical exome panel may not cover all crucial genes or areas, and variation categorization might be complex and subjective. Further exome sequencing may overlook regulatory regions and noncoding variations that may play a role in illness, and variant identification methods may yield false positives or false negatives. Using ClinVar alone to evaluate variants has limits, and prediction methods may not always adequately represent in vivo biological importance. Finally, as monogenic disorders are rare and poorly understood, it hints at a general restriction in our understanding of rare diseases. Because the CONVEX pipeline is considered a consensus variant pipeline, it is critical to confirm its performance by comparing it with known standards and datasets [17]. As genomics is a dynamic field, new genes and variant classifications might develop, affecting the relevance and accuracy of the study findings over time. In conclusion, this research lays the groundwork for further explorations into the genetic landscape of rare neurological disorders in the Indian population, paving the path for improved diagnosis, treatment, and, ultimately, a brighter future for those affected by these debilitating conditions.