Introduction
Autism spectrum disorder (ASD) encompasses a group of developmental impairments that impact how a person perceives and socialises with others. It shows impairments in communication, social interaction and stereotyped behaviours (Diagnostic and Statistical Manual of Mental Disorders, IV and V).1,2 ASD has also been found to be associated with many neurological disorders with varying degrees of severity.3 Results from various genomic screens implicated the association of many genes with ASD.4–6 Autism databases revealed that around 1,237 genes were found to be associated with ASD (https://gene.sfari.org/).7 Among them, many genes encoding cell adhesion molecules (CAMs) were found to play crucial roles in ASD.7 CAMs are cell surface glycoproteins that are required to play important roles in neural development, especially in communication, synaptic formation and function.8–10
The neuronal cell adhesion molecule (NrCAM) gene is a member of the immunoglobulin superfamily (IgSF), belonging to the class-II molecule and is closely related to L1CAM, CHL1 and neurofascin. Structurally, NrCAM has an extracellular region of six immunoglobulin domains, five fibronectin type III domains, a transmembrane-spanning region and a cytoplasmic intracellular region.11–13 It is located on the 7th chromosome at 7q31.1 and consists of 47 exons (NCBI Gene ID: 4897, updated on 9 September 2025).14 It is highly expressed in the brain, including the floor plate (NCBI Gene ID: 4897).15, 16NrCAM interacts with molecules, both on the same membrane (in cis) and on opposing membranes (in trans) and thus serves as both ligand and receptor for several different neuronal recognition molecules.15 As a neuronal receptor, the dynamic changes in NrCAM expression can modulate axonal growth and guidance during development in several different regions, including the spinal cord, the visual system and cerebellum in mice.17NrCAM protein promotes signalling during axonal growth.18
Allelic variants of this gene have been associated with autism.13, 18, 19 Thus, NrCAM plays a significant role in the development of the autistic brain. In this study, genetic variations in the NrCAM gene in individuals with ASD were analysed to find out the association with ASD. Also, the present study investigates this association for the first time in Indian autistic individuals.
Methods
Subjects
The subjects included in this study were from four districts (Shivamogga, Chikmagaluru, Davanagere and Haveri) of Karnataka state. We identified 49 individuals who fulfilled the DSM-IV diagnostic criteria for ASD. As the individuals with ASD were unable to self-report, questionnaires were prepared for the parents/caretakers and written consent was obtained from them.
Severely affected individuals were selected for the genotyping.
Genomic DNA Extraction, Polymerase Chain Reaction and Sequencing
Genomic deoxyribonucleic acid (DNA) was isolated from the peripheral blood sample using the ‘QIAamp DNA blood mini kit’ (QIAGEN, Germany). Specific target primers (Table 1) were designed, and the specific exons were amplified by polymerase chain reaction (PCR). The PCR-amplified products were sequenced for both forward and reverse strands by Sanger sequencing.
Sequencing was carried out using an Applied Biosystems automated sequencer (ABI 3730xl). NrCAM exon sequences of individuals with ASD were matched with the reference human genome (NCBI Gene ID 4897) and control samples.
In Silico Analysis
The obtained sequences were analysed using the Sequencher 5.4.6 programme. SNPs were detected by aligning both the forward and reverse strands along with NCBI reference sequences. Haploview 4.2 was used for the genotype association study.
Results
In the present study, 10 exons were sequenced and 4 SNPs were identified: two in the coding region and two in the UTR (Figure 1 and Table 2). Exons were selected from each domain region of the NrCAM gene to identify the single nucleotide variations. Exon 7 has a synonymous variation in which the third base of the codon was changed from C to T [transition mutation such as AAC > AAT (both code for asparagine)].
Exon 21 has a synonymous variation in which the third base was changed from G to C [transversion mutation such as GCG > GCC (both code for alanine)]. The in silico analysis of linkage disequilibrium (LD) (D′ value between pairwise SNPs) revealed that among the four SNPs, a pair of SNPs (rs1269634 and rs2072546) has strong LD. The LD score is significant between rs1269634 and rs401433/rs449077 (Table 3).
The LD plot represents the graphical view of the NrCAM gene region (Figure 2), suggesting that NrCAM is associated with ASD.
Discussion
The NrCAM gene is expressed in neural tissue as well as non-neural tissue. It is an ankyrin-binding protein playing an important role in cell–cell communication. Earlier, allelic variations of this gene have been associated with ASD and addictive behaviour.18, 20, 21 The present study also suggests there is an association between ASD and NrCAM gene variants.
We analysed four NrCAM gene SNPs (rs1269634, rs401433, rs449077 and rs2072546) from UTR and coding regions to investigate the association between these changes and ASD. Among these four SNPs, the frequency of the T > C (rs1269634) genotype was higher in ASD than in controls. It is a putative regulatory SNP (T > C in 5′UTR of the NrCAM gene) that reveals a pivotal role in signal peptide alternative splicing.
Multiple variants of this gene may be functionally involved in CAM pathways, suggesting that this is a potentially important variant.22 In the present study, the rs1269634 in the 5′UTR showed a significant association (0.999) with the other three SNPs (rs2072546, rs401433 and rs449077). Similarly, a previous study21 has also shown that the rs1269634 and rs2072546 SNPs have a significant association with personality traits and addictive behaviour in methamphetamine abuse. This strong association between these SNPs and ASD may be high due to alterations in the biological function of the NrCAM protein, as cell–cell interactions occur in this region.
As the NrCAM protein is involved in neuron–neuron adhesion and promotes signalling during axon growth, it suggests that any variation in the NrCAM gene will affect neuronal development and cause many behavioural disorders such as ASD and personality traits.
Conclusion
Our study indicates that the allelic variant of the NrCAM gene is associated with ASD. It is revealed that these SNPs show a statistically significant association and may cause harmful effects if there were no synonymous variations. Hence, it is necessary to screen the complete NrCAM gene to find out the NrCAM gene variants and their possible association with ASD.