Abstract
Background
Autism spectrum disorder (ASD) is the most complex neurodevelopmental disorder with impairments in communication, social interaction and stereotyped behaviours. ASD has been found to increase immeasurably in India, with estimates ranging from 1 in 68 to 1 in 100 children. Genetically, more than 1,000 genes have been linked with ASD. Among them, the neuronal cell adhesion molecule (NrCAM) gene is one of the candidate genes and is important for its critical role in the development and function of the nervous system, particularly in wiring the brain through axon guidance and organising myelinated nerve structures. Disruptions in this gene are associated with various neurodevelopmental, psychiatric and cancer-related disorders.
Purpose
The present study aimed to understand the association of the NrCAM gene with ASD.
Methods
Ten exons of the NrCAM gene (one from 5′UTR, five from the immunoglobulin domain, three from the fibronectin domains and one from 3′UTR region) were selected and screened for single nucleotide polymorphisms (SNPs) by Sanger sequencing. Several in silico analyses were used for the association study.
Results
The results revealed four SNPs from coding and untranslated regions (rs2072546, rs401433, rs1269634 and rs449077) of the NrCAM gene which were analysed for association by linkage disequilibrium analysis.
Conclusion
The present study clearly suggests that these SNPs of the NrCAM gene are associated with ASD.
Keywords: Autism spectrum disorder, NrCAM gene, single nucleotide polymorphism, linkage disequilibrium
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, 16 NrCAM 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. 17 NrCAM 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.
Table 1. Sequences of Primers and Exons Used in Polymerase Chain Reaction.
| Sl. No. | Primers | Exons | Region/Domain |
| 1 | F′-CACGGCTAGTTCTCTGTGCTGCAG R′-CAGACACCTTCTCCATTCACGCAG |
4 | 5′UTR |
| 2 | F′-AGATATGAGGGCTTATTGCTCTC R′-ATGTCACAGAAGCTGAGTCTTGGA |
7 | Ig |
| 3 | F′-AGCTGTGGAATCACTGACTG R′-CCTATCTCCTCCCACTGCAAAGA |
8 | Ig |
| 4 | F′-TGTCAGGGAAGTAGAGTTAGAGTT R′-TGGCTTCCTGATAATGAACCTG |
9 | Ig |
| 5 | F′-AGGTAACAGATTCACACTCACTTTG R′-ACCTGATACCTTCTTGGGTGCAT |
16–17 | Ig |
| 6 | F′-GGTTGCTTGTGTTGGGTTGC R′-ACTCCCATCATACAGCTGCC |
21–22 | Fn |
| 7 | F′-TGGGTCTCGTGCTGTTTTCA R′-GTAGCCCTTCTGTCAGGCAAAACC |
25 | Fn |
| 8 | F′-AACTCATCCTCGTCATTGGAG R′-ATTGAACTGTATCCTGCAGAGGA |
34 | 3′ UTR |
| 9 | F′-GAACTGGAATGCAAAGTGCATAC R′-CATCATGTGCTTCAGTTGGCAAC |
||
| 10 | F′-GCAGTGAATGGGATTGGAAAGCATC R′-CCTGTAGTGTCCCTCTTCAGACA |
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.
Figure 1. Protein Domain Model of NrCAM Gene Showing the Location of Single Nucleotide Polymorphisms.
Source: The protein domain model was adapted from Ishiguro et al. (2006)11 and the SNPs identified in the present study are indicated on the model).
Table 2. Genotypic Association of the Neuronal Cell Adhesion Molecule (NrCAM) Gene in Autism Spectrum Disorder.
| SNP Name | Description of SNP | Exon Location | Domain | Type of Variant |
| rs449077 | A > G | 34 | 3′UTR | 3′UTR |
| rs401433 | G > C | 21 | Fn-III | Coding/synonymous |
| rs2072546 | C > T | 7 | Ig | Coding/synonymous |
| rs1269634 | T > C | 4 | 5′UTR | 5′UTR |
Note: SNP: Single nucleotide polymorphism.
Table 3. Linkage Disequilibrium (D′) Between Neuronal Cell Adhesion Molecule Gene Single Nucleotide Polymorphisms.
| rs449077 | rs401433 | rs2072546 | rs1269634 | |
| rs1269634 | 0.9994 | 0.9994 | 0.9996 | – |
| rs2072546 | 0.9988 | 0.9988 | – | |
| rs401433 | 0.0625 | – | ||
| rs449077 | – |
Figure 2. Linkage Disequilibrium (LD) Plot of the Neuronal Cell Adhesion Molecule Gene. Regions with Strong LD and LOD (D′ = 1 and LOD > 2) Scores are Shown in Bright Red. Blocks with Shades of Blue and White Show a Decrease in D′ value and LOD.
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 study 21 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.
Acknowledgements
The authors are thankful to the Science and Engineering Research Board, Department of Science and Technology (DST-SERB), Government of India, New Delhi, for financial support and to the Kuvempu University for providing the facilities. The authors are grateful to all the study participants (autistic children), their parents and caretakers. The authors also thank the doctors and management of special schools.
The authors declared no potential conflicts of interest with respect to the research, authorship and publication of this article.
Funding: The authors disclosed receipt of the following financial support for the research, authorship and/or publication of this article: This study was supported by the Science and Engineering Research Board, Department of Science and Technology (SB/EMEQ-128/2014), Government of India, New Delhi.
ORCID iDs: Elumalai Chandrakala 
https://orcid.org/0000-0002-6001-9825
Kakanahalli Nagaraj 
https://orcid.org/0000-0002-3516-0560
Authors’ Contributions
The study was designed and conceptualised by Dr Kakanahalli Nagaraj. He supervised each step of the work, criticised the manuscript and interpreted the results. Dr Elumalai Chandrakala collected the samples of ASD individuals, conducted experiments, analysed and interpreted the results and prepared the manuscript. Both authors read and approved the final manuscript.
Statement of Ethics
The present study was approved by the Institutional Ethics Committee of the Kuvempu University (Registration number KU/IEC/05-10/2014-15), Shivamogga, Karnataka, India.
Informed Consent
Written informed consent was obtained from all participants prior to their inclusion in the study. Participants were informed about the purpose and procedures of the study, and confidentiality and anonymity of their personal information were maintained.
References
- 1.American Psychiatric Association. Autistic disorder. In: Diagnostic and Statistical Manual of Mental Disorders. 4th ed. Washington, DC: American Psychiatric Association; 1994. [Google Scholar]
- 2.American Psychiatric Association. Autism spectrum disorder. In: Diagnostic and Statistical Manual of Mental Disorders. 5th ed. Washington, DC: American Psychiatric Association; 2013. [Google Scholar]
- 3.Chandrakala E and Nagaraj K.. Analysis of co-occurrence of autism spectrum disorder with other neurological disorders. Asian J Pharm Clin Res 2020; 13(5): 69–72. [Google Scholar]
- 4.Jiang Y, Yuen RKC, Jin X, et al. Detection of clinically relevant genetic variants in autism spectrum disorder by whole-genome sequencing. Am J Hum Genet 2013; 93: 1–15. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Losh M, Sullivan PF, Franz CP, et al. Current developments in the genetics of autism: from phenome to genome. J Neuropathol Exp Neurol 2009; 67(9): 829–837. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Wall DP, Esteban FJ, Deluca TF, et al. Comparative analysis of neurological disorders focuses genome-wide search for autism genes. Genomics 2009; 93(2): 120–129. [DOI] [PubMed] [Google Scholar]
- 7.Simons Foundation Autism Research Initiative. SFARI Gene [Internet]. New York: Simons Foundation; [cited 11 September 2025]. Available from: https://gene.sfari.org/ [Google Scholar]
- 8.Ye H, Liu J, and Wu JY.. Cell adhesion molecules and their involvement in autism spectrum disorder. Neurosignals 2011; 18(2): 62–71. [DOI] [PubMed] [Google Scholar]
- 9.Betancur C, Sakurai T, and Buxbaum JD.. The emerging role of synaptic cell-adhesion pathways in the pathogenesis of autism spectrum disorders. Trends Neurosci 2009; 32(7): 402–412. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Zhong X, Drgonova J, Li C, et al. Human cell adhesion molecules: annotated functional subtypes and overrepresentation of addiction-associated genes. Ann N Y Acad Sci 2015; 1349(1): 83–95. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Ishiguro H, Liu QR, Gong JP, et al. NrCAM in addiction vulnerability: positional cloning, drug-regulation, haplotype-specific expression, and altered drug reward in knockout mice. Neuropsychopharmacology 2006; 31(3): 572–584. [DOI] [PubMed] [Google Scholar]
- 12.Burden-Gulley SM and Lemmon V.. Ig superfamily adhesion molecules in the vertebrate nervous system: binding partners and signal transduction during axon growth. Semin Dev Biol 1995; 6(2): 79–87. [Google Scholar]
- 13.Hortsch M. Structural and functional evolution of the L1 family: are four adhesion molecules better than one? Mol Cell Neurosci 2000; 15(1): 1–10. [DOI] [PubMed] [Google Scholar]
- 14.National Center for Biotechnology Information. NRCAM gene [Internet]. Bethesda (MD): National Library of Medicine; [cited 11 September 2025]. Available from: https://www.ncbi.nlm.nih.gov/gene/4897 [Google Scholar]
- 15.Sakurai T. The role of NrCAM in neural development and disorders—beyond a simple glue in the brain. Mol Cell Neurosci 2012; 49(3): 351–363. [DOI] [PubMed] [Google Scholar]
- 16.Wang B, Williams H, Du J, et al. Alternative splicing of human NrCAM in neural and nonneural tissues. Mol Cell Neurosci 1998; 10: 287–295. [DOI] [PubMed] [Google Scholar]
- 17.Lustig M, Erskine L, Mason CA, et al. Nr-CAM expression in the developing mouse nervous system: ventral midline structures, specific fiber tracts, and neuropilar regions. J Comp Neurol 2001; 434(1): 13–28. [DOI] [PubMed] [Google Scholar]
- 18.Bonora E, Lamb JA, Barnby G, et al. Mutation screening and association analysis of six candidate genes for autism on chromosome 7q. Eur J Hum Genet 2005; 13(12): 198–207. [DOI] [PubMed] [Google Scholar]
- 19.Sakurai T, Ramoz N, Reichert JG, et al. Association analysis of the NrCAM gene in autism and in subsets of families with severe obsessive-compulsive or self-stimulatory behaviors. Psychiatr Genet 2006; 16(6), 251–257. [DOI] [PubMed] [Google Scholar]
- 20.Marui T, Funatogawa I, Koishi S, et al. Association of the neuronal cell adhesion molecule (NRCAM) gene variants with autism. Int J Neuropsychopharmacol 2008; 12(1): 1–10. [DOI] [PubMed] [Google Scholar]
- 21.Yoo BK, Shim JC, Lee BD, et al. Association of the Neuronal Cell Adhesion Molecule (NrCAM) gene variants with personality traits and addictive symptoms in methamphetamine use disorder. Psychiatry Investig 2012; 9: 400–407. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Shirzad H, Beiraghi N, Kachoui M, et al. Family-based whole-exome sequencing for identifying novel variants in consanguineous families with schizophrenia. Iran Red Crescent Med J 2017; 19(2): e35788. [Google Scholar]