Analysis of epigenetic mutation of AGTR2 gene responsible for Autism across three generation: An exploratory study

Analysis of epigenetic mutation of AGTR2 gene responsible for Autism across three generation: An exploratory study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Analysis of epigenetic mutation of AGTR2 gene responsible for Autism across three generation: An exploratory study Priya Misra, Leena Bhardwaj, Vijay Kumar, Kamal Chauhan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7040442/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Autism Spectrum Disorder (ASD) is a complex neurodevelopmental condition with multifactorial unknown etiology that involves epigenetic influences. The epigenetic refers to the impact of environmental conditions on the nucleotide sequence. This exploratory study investigated the role of mutations in the AGTR2 gene, which encodes angiotensin II receptor type 2, in ASD pathogenesis. Six children with ASD participated in this study and their base pair nucleotide sequence was compared with the maternal relatives across three familial generations. Saliva samples were collected using swabbing technique, as the source for DNA extraction. Sanger sequencing was used to identify genetic mutations, especially the frameshift and point mutations in base pair nucleotide sequence of AGTR2 across three generations. Results revealed that the recurrent frameshift mutations in ASD affected individuals and their close relatives at 1-77 base pair length, suggesting potential heritable transmission. Point mutations were also observed although, their locations varied between individuals, indicating a possible contribution of de novo mutations. Consistently high adenine-thymine (A=T) content was found across all samples, suggesting genomic instability, which may act as a hotspot locus of mutation. Epigenetic mechanisms, such as DNA methylation and DNA Acetylation influenced by stressful environmental exposures were considered as the prime factor for the intergenerational inheritance of ASD susceptibility. These findings support the hypothesis that both inherited and environmental factors contribute to ASD risk, with AGTR2 emerging as a significant candidate gene for understanding complex etiology of ASD and supports the potential for AGTR2 related biomarkers in diagnosis and early intervention strategies. Angiotensin II AGTR2 Autism Neurodevelopmental disorder Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Autism spectrum disorder (ASD) is a multifactorial neurodevelopmental condition characterized by deficits in social communication and interaction, accompanied with certain behavioral pattern such as restricted, repetitive patterns of behavior and loss of interests [1]. According to a survey conducted by the World Health Organization (2020), ASD affects approximately 1 in 160 children globally. The rate of prevalence in male child is observed to be four times higher than the female child. as reported by the U.S. Centers for Disease Control and Prevention [2]. Although the exact etiology of ASD remains elusive, twin and family studies have established a strong genetic component, with concordance rates exceeding 70% in monozygotic twins and an increased prevalence among siblings [3]. Numerous genes have been implicated in this condition, including IMMP2L , DOCK4 , GRIK1 , RELN , CNTNAP2 , and MET , all of which play roles in synaptic function, neural connectivity, and gene regulation [4]. Among these, the AGTR2 gene has recently emerged as a potential contributor, although its role in ASD remains under active investigation [5]. AGTR2 is located at Xq23 consisting of three exons. ATGR2 gene encodes angiotensin II receptor type 2, a protein involved in neural, renal, and vascular development. Mutations in this gene, particularly frameshift and point mutations, can result in dysfunctional receptors, disrupting neurodevelopmental processes critical to cognition and behavior. Frameshift mutations typically arise from nucleotide insertions or deletions that are not divisible by three, which alter the gene’s reading frame and lead to truncated, or nonfunctional protein products. In addition to these genetic disruptions, emerging studies have highlighted the influence of epigenetic modifications such as DNA methylation and histone modulations on AGTR2 gene expression [6]. These modulations do not alter the DNA sequence itself but can silence or activate genes, potentially contributing to ASD phenotypes. Abusive habits like smoking and alcohol consumption during pregnancy, viral infections, and nutritional deficiencies, or exposure to stressful environmental conditions like regular domestic violence supposed to act as mutagenic agent to bring substantial intrauterine epigenetic changes which could be the risk factors that leads to range of autistic behaviour.in children. [7]. Recent research also supports the concept of transgenerational epigenetic inheritance in ASD, where environmental exposure can produce heritable epigenetic marks. in rodent models, prenatal exposure to valproic acid has been shown to induce autism-like traits in offspring, which are epigenetically transmitted to at least the third generation [8]. This evidence underscores the importance of considering both genetic and non-genetic factors in ASD research. The multigenic and multifactorial nature of ASD suggests that the interaction between genetic predisposition and epigenetic regulation is crucial for understanding its heritability and clinical variability [9, 10]. Therefore, the present study aimed to investigate the role of epigenetic mutation of AGTR2 gene that has the potential factor to cause range of ASD. Objective Thus, the primary objective of the study was to identify the mutant loci in the nucleotide strand of AGTR2 gene sequence; that is heritable to forthcoming generation and the secondarily to analyse the pattern of inheritance of mutant loci into the generations. Methods This study involved six children diagnosed with autism spectrum disorder (ASD) and their biological family members. An ethical approval for the study was obtained from the Institutional Ethical Committee (Ref # IEC-AMS/AUH/75/2024-2025). Prior to participation, written informed consent was obtained from the parents of each child with ASD. Participant identification was performed using the Childhood Autism Rating Scale (CARS), a widely recognized diagnostic tool for assessing ASD severity [11]. The inclusion criteria required that the children be aged between 5 to 7 years, have mild to moderate ASD symptoms, and exhibit behavioral issues. Furthermore, only biological mothers and grandmothers were included in this study to maintain genetic consistency. Children with severe ASD and families with stepmothers or step-grandmothers were excluded. The study followed an ex-post facto design, and participants were selected using a non-random sampling method. Genetic analysis was conducted using a multi-step protocol, beginning with the collection of saliva samples using swabbing technique from the ASD-affected children, their mothers, and grandmothers [12, 13]. The samples were stored at <0°C temperatures to prevent fungal contamination. DNA isolation was carried out using an organic extraction method as per the protocol followed in Biology Division, Directorate of Forensic Science New Delhi [14]. Proteinase K, phosphate buffer, and DTT were added to each sample, followed by centrifugation and 1000 RPM, for fifteen minutes. The mixture was incubated overnight at 56°C. Subsequently, organic solvents such as alcohol, chloroform, and phenol were added, and the samples were centrifuged at 7000 RPM for 30 min. The resulting pellet was resuspended in ethanol and centrifuged again, after which it was dissolved in a buffer containing Tris base, EDTA, and distilled water (pH 8). High-purity DNA was isolated following final centrifugation from the pellet. The extracted DNA was analysed for its quality by electrophoresis technique and visualized by X-ray illuminator. Refer to Figure 1. Subsequent purifications were carried out at ATSBIO Scientific Private Limited, including Polymerase Chain Reaction (PCR), primer synthesis, and Sanger sequencing. PCR was used to amplify specific DNA sequences using DNA polymerase, facilitating downstream genetic analysis. Primer synthesis involved short, specific DNA fragments that serve as initiation points for DNA replication. Sanger sequencing was used to determine the nucleotide sequence of the amplified DNA, enabling the specific identification of potential genetic variants. Instruments used throughout the process included a vortex mixer (for homogenizing reagents), Centricon centrifugal device (for DNA isolation under controlled speed and temperature), gel electrophoresis apparatus (for separating DNA fragments by size and charge), and UV-ray transilluminator (for visualizing DNA bands post-electrophoresis on agarose gels). These combined methodologies ensured the reliability and accuracy of the genetic data obtained in study. Results The AGTR2 gene was analysed among the rural populations of Haryana. The samples of children include moderate to severe degree of Autism. High quality DNA extracted from saliva is a clinically validated method for DNA extraction. The primer for AGTR2 gene was identified rs121917810. The primer designed for AGTR2 gene 3’-CGTGACCAAGTCCTGAAGATGG-5’ and 5’-GGAAGTGCCAGGTCAATGAGTG-3’. The Sanger sequencing was performed on 22 samples. Interpretation of Sanger sequencing and comparison of DNA sequence with their respective family member’s samples were conducted at 4 different levels. Coding for each sample was done. Refer to Table 1 . Table 1 Labelling of sample for ASD child, siblings, mother, father, paternal grand father Pairs Labelled sample Family members 3-G Pair 3G 1 ASD child 3G 1 S Sister 3G 2 M Mother 3G 3 P Paternal grandfather 2-G Pair 2G 1 ASD Child 2G 2 M Mother 1-G Pair 1G 1 ASD Child 1G 1 S Brother I-G Pair 1IG 1 ASD child (Independent) 2IG 1 ASD child (Independent) 3IG 1 ASD child (Independent) 3-G Pair In the 3-G pair, a genetic analysis was conducted involving an ASD child, both parents, a sibling, and the grandfather. The focus was on the AGTR2 gene, specifically the 1–77 forward sequence of nitrogenous bases. A frameshift mutation was identified in this region was present in the child, sibling, and mother. However, this mutation was not observed at the same location in the grandfather. This observation suggests that the mutations are unlikely inherited from the paternal grandfather's lineage. Point mutations and translocations have been observed in multiple family members, including children with ASD. However, the specific location (nucleotide or gene) of the mutation differs between children with ASD and their relatives. This suggests genetic heterogeneity within the family. The consistent presence of the mutation in immediate family members indicates a possible inherited or de novo mutation from the mother. Refer to Fig. 2 . 2-G Pair In the 2-G pair, a frameshift mutation was identified in both the ASD-affected child and their biological mother, specifically spanning nucleotide positions 1 to 60. The presence of this identical frameshift variant in both individuals suggests a maternally inherited mutation that may have contributed to ASD susceptibility in this case. In addition to the shared frameshift variant, both the child and mother exhibited point mutations and translocations. However, these point mutations and translocations were located at different nucleotide positions in everyone, indicating that they are independent mutational events rather than inherited variants. The lack of positional overlap in point mutations suggests that they may represent either de novo changes or background genetic variation. Refer to Fig. 3 . 1-G Pair In the 1-G pair, both the ASD-affected child and their unaffected sibling were found to carry an identical frameshift mutation spanning nucleotide positions 1 to 67. The presence of this shared mutation in both siblings suggests a potential germline variant inherited from a common parent, which may contribute variably to the ASD phenotype, depending on its functional impact and penetrance. Like the findings in the above groups, point mutations were also observed in both individuals; however, these mutations occurred at distinct nucleotide positions, indicating that they are not shared and may represent individual-specific variants. The differing locations of the point mutations suggest possible de novo occurrences or individual genetic variations, warranting further investigation to assess their functional relevance. Refer to Fig. 4 . I-G Pair In the I-G pair, sequencing of all independent children with ASD samples revealed consistent presence of frameshifts, point mutations, and translocations. These genetic alterations were identified within short nucleotide sequences ranging from 1 to 80 bps in length. The coexistence of these types of mutations suggests a potential synergistic effect on gene disruption and may indicate a critical mutational hotspot contributing to the molecular pathology observed in this subgroup. Refer to Fig. 5 . Analysis across the four study groups revealed the consistent presence of frameshift mutations in both ASD-affected individuals and their respective family members. This suggests a potential role for inherited frameshift variants in ASD susceptibility. In addition to frameshift mutations, point mutations were detected in all analyzed individuals. However, these point mutations occur at distinct genomic locations in each case, indicating individual-specific variations rather than a single inherited variant. Furthermore, which involve abnormal repositioning of nucleotide sequences from one genomic region to another, may disrupt critical gene regulatory elements or coding regions, potentially altering gene expression profiles essential for neurodevelopment. Additionally, sequence analysis revealed consistently high A = T content in all affected individuals and their family members. Enrichment of adenine-thymine base pairs may have implications for genome stability, gene regulation, or mutational hotspots that contribute to ASD pathogenesis. These findings underscore the complex genetic landscape of ASD, characterized by a combination of shared and unique mutations, as well as potential sequence composition biases that warrant further investigation. Discussion The findings of this study highlight the recurrent presence of frameshifts and point mutations among individuals with ASD and their family members across three independent groups. Notably, the shared frameshift mutations observed in Groups 1, 2, and 3 between the ASD-affected individuals and their respective relatives suggest a possible inherited component contributing to ASD susceptibility. This observation aligns with previous studies that have reported two specific mutations in the AGTR2 gene have been identified: a 62G→T transversion (G21V) and a 157A→T transversion (I53F), both associated with severe mental retardation and developmental delays [ 15 ]. Frameshift mutations, such as the deletion of thymine within a string of eight Ts, have been identified in patients with mental retardation, some of whom exhibit autistic behaviors [ 16 ]. These mutations can lead to significant changes in protein structure, which potentially disrupting brain development and function [ 17 ]. Although the primary focus of AGTR2 mutations has been on mental retardation, some patients with these mutations also display symptoms consistent with ASD, such as pervasive developmental disorders [ 18 ]. The overlap in symptoms suggests a potential link between AGTR2 mutations and ASD, although direct evidence specifically connects AGTR2 frameshift mutations to ASD [ 19 ]. In contrast, the identification of distinct point mutations at different genomic positions within each family supports the role of individual-specific or de novo mutational events, consistent with earlier genomic studies that emphasized the importance of de novo single nucleotide variants (SNVs) in ASD pathogenesis [ 20 , 21 ]. Previous studies indicates that mutations in AGTR2 , such as point mutations, are associated with severe mental retardation and pervasive developmental disorders that can manifest in children [ 18 ]. Studies have shown that multiple inherited variants in ASD-associated genes, including AGTR2, can contribute to the pathogenesis of the disorder, particularly in families with multiple affected siblings [ 22 ]. Paternally inherited structural variants, particularly in non-coding regions, have been found to be preferentially transmitted to affected offspring, indicating a significant paternal contribution to ASD risk [ 23 ]. The coexistence of shared unique point mutations suggests a multifactorial genetic basis, where both inherited and spontaneous mutations interact to influence the phenotypic outcomes. Interestingly, our observation of consistently high A = T content across the examined sequences is noteworthy. Regions rich in adenine-thymine base pairs have been previously associated with increased genomic instability, replication errors, and the formation of secondary DNA structures that may predispose to frameshift or point mutations [ 24 , 25 ]. This compositional bias may act as a mutational hotspot, thereby increasing the likelihood of pathogenic mutations in functionally critical genes neurodevelopment-related genes. Environmental factors such as maternal smoking, exposure to pollutants, and stress during gestation have been linked to altered DNA methylation patterns, which may increase the risk of ASD [ 26 ]. Research indicates that not only parental but also grandparental gene is influenced with DNA methylation, exposure can influence DNA methylation, suggesting a multigenerational aspect of ASD risk [ 27 ]. Histone modulations i.e. DNA methylation and DNA Acetylation can transmits from generation to generations, affecting gene expression in subsequent generations, which may contribute to the prevalence of ASD in families[ 27 ]. Davies et al. (2012) also showed some that sites of methylation variation across individuals, potentially influenced by genetic or environmental factors, persist across blood and brain tissues [ 28 ]. In the current study, a genetic variant in the angiotensin II receptor type 2 (AGTR2) gene was identified in association with ASD, consistent with findings from previous research. Previous studies have suggested that AGTR2 plays a crucial role in regulating uteroplacental circulation, which is sensitive to various vasoactive agents such as angiotensin II and oxytocin (OXT), both of which increase vascular resistance which activates sympathetic nervous system [ 19 ]. Notably, AGTR2 has been implicated in the modulation of blood levels of arginine vasopressin (AVP) and OXT neuropeptides that are closely associated with the regulation of social behavior [ 29 ]. Furthermore, angiotensin II (Ang II), the key effector peptide of the renin-angiotensin system, is believed to interact with central nervous system neurotransmitters, including dopamine and serotonin. These interactions suggest a plausible mechanism by which Ang II influences behavioral and cognitive processes [ 30 ]. Studies have also proposed that Ang II in the brain may induce dopaminergic neuronal death through reactive oxygen species generation, contribute to oxidative stress and neurodegeneration [ 31 ]. In addition to dopamine depletion, subsequent neuroinflammatory responses are believed to play a role in the pathophysiology of autism and other neurodevelopmental disorders [ 32 ]. Ang II, known for its pro-inflammatory effects, mediates its physiological functions primarily through two receptor subtypes: angiotensin II type 1 (AT1) and type 2 (AT2) receptors. Both receptors are widely distributed across various brain regions, including those implicated in cognitive and behavioral regulation [ 33 ]. The presence of AGTR2 in these areas supports the hypothesis that dysregulation of the RAS pathway contributes to ASD development. Future Directions Although the present study relates with common mendelian principle of inheritance in heterozygous and homozygous conditions particularly directs towards the pedigree analysis in population genetics. However, major demand for pedigree analysis with larger ASD population needs further exploration. The present study may also be elaborated by involving paternal and maternal pedigree of ASD individuals across different age groups. Identification of specific epigenetic factors causing ASD; could be helpful in taking preventive measures in the incidence of ASD. Since the key autism symptoms do not confine with the epigenesis into the AGTR2 gene only, it should be further explored in the loci of other gene i.e. ATRX, PQBP1, SLC16A2 gene sequence also; that is responsible to cause several associated symptoms in ASD. Conclusion Studies have identified specific differentially methylated genes associated with ASD, indicating their potential as diagnostic biomarkers [ 34 ]. Peripheral DNA methylation analysis in children with idiopathic ASD shows promise for clinical application, although further research is needed [ 35 ]. Our findings support the growing body of evidence that ASD is characterized by genetic heterogeneity involving both inherited and non-inherited mutations. The presence of shared frameshift mutations among affected and unaffected relatives raises questions regarding penetrance and the potential role of modifier genes or environmental factors in influencing phenotypic expression. Furthermore, the observation of high A = T regions warrant deeper investigation into sequence-specific mutational mechanisms and their functional consequences in ASD. Future studies integrating whole-genome sequencing, functional assays, and epigenetic profiling are necessary to delineate the contribution of these variants to ASD and explore their potential as biomarkers for early diagnosis or therapeutic targeting. Statements and Declarations Funding: The authors declare that no financial assistance/grants were received during the preparation of this manuscript. Conflict of Interest: The authors of this manuscript declare that no competing interest exists. Author Contributions : Study conception and design : PM, LB, VK; Material preparation, data collection and analysis: PM, LB, VK, KC; Preparation of first draft of the manuscript: PM, LB, VK, KC; Proof reading and Final approval of the manuscript: PM, LB, VK, KC. Ethics approval: Yes, Ref # IEC-AMS/AUH/75/2024-2025 Consent to participate: Informed written consent was obtained from each participants/parent. Consent to Publish: Yes, informed written consent was obtained from each participant/parent. References Bertelli, M.O., et al., Autism spectrum disorder , in Textbook of psychiatry for intellectual disability and autism spectrum disorder . 2022, Springer. p. 369-455. 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International journal of molecular sciences, 2023. 24 (11): p. 9138. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7040442","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":489401886,"identity":"3a2ccc88-7492-4344-86c3-dfa3b64ca2fe","order_by":0,"name":"Priya Misra","email":"","orcid":"","institution":"Amity University Haryana-Gurugram Medical School","correspondingAuthor":false,"prefix":"","firstName":"Priya","middleName":"","lastName":"Misra","suffix":""},{"id":489401887,"identity":"c51b65ed-34ac-43b3-b48b-0a93601c7e89","order_by":1,"name":"Leena Bhardwaj","email":"","orcid":"","institution":"Amity University Haryana-Gurugram Amity School 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through electrophoresis\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7040442/v1/2feaf2e7c792810b81ab3916.png"},{"id":87695542,"identity":"096a067b-5f55-4e5d-8381-359f7db5e194","added_by":"auto","created_at":"2025-07-28 05:53:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":361904,"visible":true,"origin":"","legend":"\u003cp\u003eBase pair template matching for three generations (3G) for children with ASD (3G\u003csub\u003e1\u003c/sub\u003e) with their sister (3G\u003csub\u003e1\u003c/sub\u003eS), mother (3GM\u003csub\u003e1\u003c/sub\u003e) and subsequently with their paternal grandfather (3GP\u003csub\u003e1\u003c/sub\u003e)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7040442/v1/6d50e81bc55f8349bee1d67f.png"},{"id":87694440,"identity":"e881337d-1494-4e0d-a1ae-b7a8ec8825e3","added_by":"auto","created_at":"2025-07-28 05:45:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":187539,"visible":true,"origin":"","legend":"\u003cp\u003eBase pair template matching for two generations (2G) for children with ASD (2G\u003csub\u003e1\u003c/sub\u003e) with their mother (2GM\u003csub\u003e1\u003c/sub\u003e)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7040442/v1/d901a990333088303d0fdbbb.png"},{"id":87693923,"identity":"3ee87821-e520-4cc0-b352-19b2c96d6346","added_by":"auto","created_at":"2025-07-28 05:37:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":290699,"visible":true,"origin":"","legend":"\u003cp\u003eBase pair template matching of children with ASD (1G\u003csub\u003e1\u003c/sub\u003e) with their corresponding siblings (brother) (1GS\u003csub\u003e1\u003c/sub\u003e) for same or one generations (1G)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7040442/v1/baf74803ab291429569bfda9.png"},{"id":87693926,"identity":"035fac96-2c4f-4309-84ad-3e84aeb42c36","added_by":"auto","created_at":"2025-07-28 05:37:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":266422,"visible":true,"origin":"","legend":"\u003cp\u003eBase pair template matching of three independent samples of children with ASD, however sample of siblings or parents of these children were not processed. These samples were labeled as 1IG\u003csub\u003e1\u003c/sub\u003e, 2IG\u003csub\u003e1 \u003c/sub\u003eand 3IG\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7040442/v1/d49378bd84e496fd6085514f.png"},{"id":90594008,"identity":"bfde4c4d-2e86-4753-8be5-1f0d25204912","added_by":"auto","created_at":"2025-09-04 13:19:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1925027,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7040442/v1/a0b9e93e-8e08-48cb-9c70-644ffab64d5e.pdf"}],"financialInterests":"","formattedTitle":"Analysis of epigenetic mutation of AGTR2 gene responsible for Autism across three generation: An exploratory study","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAutism spectrum disorder (ASD) is a multifactorial neurodevelopmental condition characterized by deficits in social communication and interaction, accompanied with certain behavioral pattern such as restricted, repetitive patterns of behavior and loss of interests [1]. According to a survey conducted by the World Health Organization (2020), ASD affects approximately 1 in 160 children globally. The rate of prevalence in male child is observed to be four times higher than the female child. as reported by the U.S. Centers for Disease Control and Prevention [2]. Although the exact etiology of ASD remains elusive, twin and family studies have established a strong genetic component, with concordance rates exceeding 70% in monozygotic twins and an increased prevalence among siblings [3]. Numerous genes have been implicated in this condition, including \u003cem\u003eIMMP2L\u003c/em\u003e, \u003cem\u003eDOCK4\u003c/em\u003e, \u003cem\u003eGRIK1\u003c/em\u003e, \u003cem\u003eRELN\u003c/em\u003e, \u003cem\u003eCNTNAP2\u003c/em\u003e, and \u003cem\u003eMET\u003c/em\u003e, all of which play roles in synaptic function, neural connectivity, and gene regulation [4]. Among these, the \u003cem\u003eAGTR2\u003c/em\u003e gene has recently emerged as a potential contributor, although its role in ASD remains under active investigation [5].\u003c/p\u003e\n\u003cp\u003eAGTR2 is located at Xq23 consisting of three exons. \u003cem\u003eATGR2\u003c/em\u003e gene encodes angiotensin II receptor type 2, a protein involved in neural, renal, and vascular development. Mutations in this gene, particularly frameshift and point mutations, can result in dysfunctional receptors, disrupting neurodevelopmental processes critical to cognition and behavior. Frameshift mutations typically arise from nucleotide insertions or deletions that are not divisible by three, which alter the gene’s reading frame and lead to truncated, or nonfunctional protein products. In addition to these genetic disruptions, emerging studies have highlighted the influence of epigenetic modifications\u0026nbsp;such as DNA methylation and histone modulations on AGTR2 gene expression [6]. These modulations do not alter the DNA sequence itself but can silence or activate genes, potentially contributing to ASD phenotypes. Abusive habits like smoking and alcohol consumption during pregnancy, viral infections, and nutritional deficiencies, or exposure to stressful environmental conditions like regular domestic violence supposed to act as mutagenic agent to bring substantial intrauterine epigenetic changes which could be the risk factors that leads to range of autistic behaviour.in children. [7].\u003c/p\u003e\n\u003cp\u003eRecent research also supports the concept of transgenerational epigenetic inheritance in ASD, where environmental exposure can produce heritable epigenetic marks. in rodent models, prenatal exposure to valproic acid has been shown to induce autism-like traits in offspring, which are epigenetically transmitted to at least the third generation [8]. This evidence underscores the importance of considering both genetic and non-genetic factors in ASD research. The multigenic and multifactorial nature of ASD suggests that the interaction between genetic predisposition and epigenetic regulation is crucial for understanding its heritability and clinical variability [9, 10]. Therefore, the present study aimed to investigate the role of epigenetic mutation of \u003cem\u003eAGTR2\u0026nbsp;\u003c/em\u003egene that has the potential factor to cause range of ASD.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObjective\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThus, the primary objective of the study was to identify the mutant loci in the nucleotide strand of AGTR2 gene sequence; that is heritable to forthcoming generation and the secondarily to analyse the pattern of inheritance of mutant loci into the generations.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eThis study involved six children diagnosed with autism spectrum disorder (ASD) and their biological family members. An ethical approval for the study was obtained from the Institutional Ethical Committee (Ref # IEC-AMS/AUH/75/2024-2025). Prior to participation, written informed consent was obtained from the parents of each child with ASD. Participant identification was performed using the Childhood Autism Rating Scale (CARS), a widely recognized diagnostic tool for assessing ASD severity [11]. The inclusion criteria required that the children be aged between 5 to 7 years, have mild to moderate ASD symptoms, and exhibit behavioral issues. Furthermore, only biological mothers and grandmothers were included in this study to maintain genetic consistency. Children with severe ASD and families with stepmothers or step-grandmothers were excluded. The study followed an ex-post facto design, and participants were selected using a non-random sampling method.\u003c/p\u003e\n\u003cp\u003eGenetic analysis was conducted using a multi-step protocol, beginning with the collection of saliva samples using swabbing technique from the ASD-affected children, their mothers, and grandmothers [12, 13]. The samples were stored at \u0026lt;0\u0026deg;C temperatures to prevent fungal contamination. DNA isolation was carried out using an organic extraction method as per the protocol followed in Biology Division, Directorate of Forensic Science New Delhi [14]. Proteinase K, phosphate buffer, and DTT were added to each sample, followed by centrifugation and 1000 RPM, for fifteen minutes. The mixture was incubated overnight at 56\u0026deg;C. Subsequently, organic solvents such as alcohol, chloroform, and phenol were added, and the samples were centrifuged at 7000 RPM for 30 min. The resulting pellet was resuspended in ethanol and centrifuged again, after which it was dissolved in a buffer containing Tris base, EDTA, and distilled water (pH 8). High-purity DNA was isolated following final centrifugation from the pellet. The extracted DNA was analysed for its quality by electrophoresis technique and visualized by X-ray illuminator. \u0026nbsp;Refer to Figure 1.\u003c/p\u003e\n\u003cp\u003eSubsequent purifications were carried out at ATSBIO Scientific Private Limited, including Polymerase Chain Reaction (PCR), primer synthesis, and Sanger sequencing. PCR was used to amplify specific DNA sequences using DNA polymerase, facilitating downstream genetic analysis. Primer synthesis involved short, specific DNA fragments that serve as initiation points for DNA replication. Sanger sequencing was used to determine the nucleotide sequence of the amplified DNA, enabling the specific identification of potential genetic variants. Instruments used throughout the process included a vortex mixer (for homogenizing reagents), Centricon centrifugal device (for DNA isolation under controlled speed and temperature), gel electrophoresis apparatus (for separating DNA fragments by size and charge), and UV-ray transilluminator (for visualizing DNA bands post-electrophoresis on agarose gels). These combined methodologies ensured the reliability and accuracy of the genetic data obtained in study.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe AGTR2 gene was analysed among the rural populations of Haryana. The samples of children include moderate to severe degree of Autism. High quality DNA extracted from saliva is a clinically validated method for DNA extraction. The primer for \u003cem\u003eAGTR2\u003c/em\u003e gene was identified rs121917810. The primer designed for \u003cem\u003eAGTR2\u003c/em\u003e gene 3\u0026rsquo;-CGTGACCAAGTCCTGAAGATGG-5\u0026rsquo; and 5\u0026rsquo;-GGAAGTGCCAGGTCAATGAGTG-3\u0026rsquo;. The Sanger sequencing was performed on 22 samples. Interpretation of Sanger sequencing and comparison of DNA sequence with their respective family member\u0026rsquo;s samples were conducted at 4 different levels. Coding for each sample was done. Refer to Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eLabelling of sample for ASD child, siblings, mother, father, paternal grand father\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePairs\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLabelled sample\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFamily members\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e3-G Pair\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3G\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eASD child\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3G\u003csub\u003e1\u003c/sub\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSister\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3G\u003csub\u003e2\u003c/sub\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMother\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3G\u003csub\u003e3\u003c/sub\u003eP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePaternal grandfather\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e2-G Pair\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2G\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eASD Child\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2G\u003csub\u003e2\u003c/sub\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMother\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e1-G Pair\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1G\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eASD Child\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1G\u003csub\u003e1\u003c/sub\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBrother\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eI-G Pair\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1IG\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eASD child (Independent)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2IG\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eASD child (Independent)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3IG\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eASD child (Independent)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3-G Pair\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn the 3-G pair, a genetic analysis was conducted involving an ASD child, both parents, a sibling, and the grandfather. The focus was on the \u003cem\u003eAGTR2\u003c/em\u003e gene, specifically the 1\u0026ndash;77 forward sequence of nitrogenous bases. A frameshift mutation was identified in this region was present in the child, sibling, and mother. However, this mutation was not observed at the same location in the grandfather. This observation suggests that the mutations are unlikely inherited from the paternal grandfather's lineage. Point mutations and translocations have been observed in multiple family members, including children with ASD. However, the specific location (nucleotide or gene) of the mutation differs between children with ASD and their relatives. This suggests genetic heterogeneity within the family. The consistent presence of the mutation in immediate family members indicates a possible inherited or \u003cem\u003ede novo\u003c/em\u003e mutation from the mother. Refer to Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e2-G Pair\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn the 2-G pair, a frameshift mutation was identified in both the ASD-affected child and their biological mother, specifically spanning nucleotide positions 1 to 60. The presence of this identical frameshift variant in both individuals suggests a maternally inherited mutation that may have contributed to ASD susceptibility in this case. In addition to the shared frameshift variant, both the child and mother exhibited point mutations and translocations. However, these point mutations and translocations were located at different nucleotide positions in everyone, indicating that they are independent mutational events rather than inherited variants. The lack of positional overlap in point mutations suggests that they may represent either de novo changes or background genetic variation. Refer to Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e1-G Pair\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn the 1-G pair, both the ASD-affected child and their unaffected sibling were found to carry an identical frameshift mutation spanning nucleotide positions 1 to 67. The presence of this shared mutation in both siblings suggests a potential germline variant inherited from a common parent, which may contribute variably to the ASD phenotype, depending on its functional impact and penetrance. Like the findings in the above groups, point mutations were also observed in both individuals; however, these mutations occurred at distinct nucleotide positions, indicating that they are not shared and may represent individual-specific variants. The differing locations of the point mutations suggest possible \u003cem\u003ede novo\u003c/em\u003e occurrences or individual genetic variations, warranting further investigation to assess their functional relevance. Refer to Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eI-G Pair\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn the I-G pair, sequencing of all independent children with ASD samples revealed consistent presence of frameshifts, point mutations, and translocations. These genetic alterations were identified within short nucleotide sequences ranging from 1 to 80 bps in length. The coexistence of these types of mutations suggests a potential synergistic effect on gene disruption and may indicate a critical mutational hotspot contributing to the molecular pathology observed in this subgroup. Refer to Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAnalysis across the four study groups revealed the consistent presence of frameshift mutations in both ASD-affected individuals and their respective family members. This suggests a potential role for inherited frameshift variants in ASD susceptibility. In addition to frameshift mutations, point mutations were detected in all analyzed individuals. However, these point mutations occur at distinct genomic locations in each case, indicating individual-specific variations rather than a single inherited variant. Furthermore, which involve abnormal repositioning of nucleotide sequences from one genomic region to another, may disrupt critical gene regulatory elements or coding regions, potentially altering gene expression profiles essential for neurodevelopment. Additionally, sequence analysis revealed consistently high A\u0026thinsp;=\u0026thinsp;T content in all affected individuals and their family members. Enrichment of adenine-thymine base pairs may have implications for genome stability, gene regulation, or mutational hotspots that contribute to ASD pathogenesis. These findings underscore the complex genetic landscape of ASD, characterized by a combination of shared and unique mutations, as well as potential sequence composition biases that warrant further investigation.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe findings of this study highlight the recurrent presence of frameshifts and point mutations among individuals with ASD and their family members across three independent groups. Notably, the shared frameshift mutations observed in Groups 1, 2, and 3 between the ASD-affected individuals and their respective relatives suggest a possible inherited component contributing to ASD susceptibility. This observation aligns with previous studies that have reported two specific mutations in the \u003cem\u003eAGTR2\u003c/em\u003e gene have been identified: a 62G\u0026rarr;T transversion (G21V) and a 157A\u0026rarr;T transversion (I53F), both associated with severe mental retardation and developmental delays [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Frameshift mutations, such as the deletion of thymine within a string of eight Ts, have been identified in patients with mental retardation, some of whom exhibit autistic behaviors [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. These mutations can lead to significant changes in protein structure, which potentially disrupting brain development and function [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Although the primary focus of \u003cem\u003eAGTR2\u003c/em\u003e mutations has been on mental retardation, some patients with these mutations also display symptoms consistent with ASD, such as pervasive developmental disorders [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The overlap in symptoms suggests a potential link between AGTR2 mutations and ASD, although direct evidence specifically connects AGTR2 frameshift mutations to ASD [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn contrast, the identification of distinct point mutations at different genomic positions within each family supports the role of individual-specific or \u003cem\u003ede novo\u003c/em\u003e mutational events, consistent with earlier genomic studies that emphasized the importance of \u003cem\u003ede novo\u003c/em\u003e single nucleotide variants (SNVs) in ASD pathogenesis [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Previous studies indicates that mutations in \u003cem\u003eAGTR2\u003c/em\u003e, such as point mutations, are associated with severe mental retardation and pervasive developmental disorders that can manifest in children [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Studies have shown that multiple inherited variants in ASD-associated genes, including AGTR2, can contribute to the pathogenesis of the disorder, particularly in families with multiple affected siblings [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Paternally inherited structural variants, particularly in non-coding regions, have been found to be preferentially transmitted to affected offspring, indicating a significant paternal contribution to ASD risk [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The coexistence of shared unique point mutations suggests a multifactorial genetic basis, where both inherited and spontaneous mutations interact to influence the phenotypic outcomes.\u003c/p\u003e\u003cp\u003eInterestingly, our observation of consistently high A\u0026thinsp;=\u0026thinsp;T content across the examined sequences is noteworthy. Regions rich in adenine-thymine base pairs have been previously associated with increased genomic instability, replication errors, and the formation of secondary DNA structures that may predispose to frameshift or point mutations [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. This compositional bias may act as a mutational hotspot, thereby increasing the likelihood of pathogenic mutations in functionally critical genes neurodevelopment-related genes. Environmental factors such as maternal smoking, exposure to pollutants, and stress during gestation have been linked to altered DNA methylation patterns, which may increase the risk of ASD [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Research indicates that not only parental but also grandparental gene is influenced with DNA methylation, exposure can influence DNA methylation, suggesting a multigenerational aspect of ASD risk [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Histone modulations i.e. DNA methylation and DNA Acetylation can transmits from generation to generations, affecting gene expression in subsequent generations, which may contribute to the prevalence of ASD in families[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Davies et al. (2012) also showed some that sites of methylation variation across individuals, potentially influenced by genetic or environmental factors, persist across blood and brain tissues [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn the current study, a genetic variant in the angiotensin II receptor type 2 (AGTR2) gene was identified in association with ASD, consistent with findings from previous research. Previous studies have suggested that \u003cem\u003eAGTR2\u003c/em\u003e plays a crucial role in regulating uteroplacental circulation, which is sensitive to various vasoactive agents such as angiotensin II and oxytocin (OXT), both of which increase vascular resistance which activates sympathetic nervous system [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Notably, \u003cem\u003eAGTR2\u003c/em\u003e has been implicated in the modulation of blood levels of arginine vasopressin (AVP) and OXT neuropeptides that are closely associated with the regulation of social behavior [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Furthermore, angiotensin II (Ang II), the key effector peptide of the renin-angiotensin system, is believed to interact with central nervous system neurotransmitters, including dopamine and serotonin. These interactions suggest a plausible mechanism by which Ang II influences behavioral and cognitive processes [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Studies have also proposed that Ang II in the brain may induce dopaminergic neuronal death through reactive oxygen species generation, contribute to oxidative stress and neurodegeneration [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn addition to dopamine depletion, subsequent neuroinflammatory responses are believed to play a role in the pathophysiology of autism and other neurodevelopmental disorders [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Ang II, known for its pro-inflammatory effects, mediates its physiological functions primarily through two receptor subtypes: angiotensin II type 1 (AT1) and type 2 (AT2) receptors. Both receptors are widely distributed across various brain regions, including those implicated in cognitive and behavioral regulation [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The presence of \u003cem\u003eAGTR2\u003c/em\u003e in these areas supports the hypothesis that dysregulation of the RAS pathway contributes to ASD development.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFuture Directions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAlthough the present study relates with common mendelian principle of inheritance in heterozygous and homozygous conditions particularly directs towards the pedigree analysis in population genetics. However, major demand for pedigree analysis with larger ASD population needs further exploration. The present study may also be elaborated by involving paternal and maternal pedigree of ASD individuals across different age groups. Identification of specific epigenetic factors causing ASD; could be helpful in taking preventive measures in the incidence of ASD. Since the key autism symptoms do not confine with the epigenesis into the AGTR2 gene only, it should be further explored in the loci of other gene i.e. ATRX, PQBP1, SLC16A2 gene sequence also; that is responsible to cause several associated symptoms in ASD.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eStudies have identified specific differentially methylated genes associated with ASD, indicating their potential as diagnostic biomarkers [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Peripheral DNA methylation analysis in children with idiopathic ASD shows promise for clinical application, although further research is needed [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Our findings support the growing body of evidence that ASD is characterized by genetic heterogeneity involving both inherited and non-inherited mutations. The presence of shared frameshift mutations among affected and unaffected relatives raises questions regarding penetrance and the potential role of modifier genes or environmental factors in influencing phenotypic expression. Furthermore, the observation of high A\u0026thinsp;=\u0026thinsp;T regions warrant deeper investigation into sequence-specific mutational mechanisms and their functional consequences in ASD. Future studies integrating whole-genome sequencing, functional assays, and epigenetic profiling are necessary to delineate the contribution of these variants to ASD and explore their potential as biomarkers for early diagnosis or therapeutic targeting.\u003c/p\u003e"},{"header":"Statements and Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e The authors declare that no financial assistance/grants were received during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest:\u003c/strong\u003e The authors of this manuscript declare that no competing interest exists.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e: \u003cstrong\u003eStudy conception and design\u003c/strong\u003e: PM, LB, VK; \u003cstrong\u003eMaterial preparation, data collection and analysis: PM, LB, VK, KC;\u003c/strong\u003e \u003cstrong\u003ePreparation of first draft of the manuscript:\u0026nbsp;\u003c/strong\u003ePM, LB, VK, KC;\u003cstrong\u003e\u0026nbsp;Proof reading and Final approval of the manuscript:\u003c/strong\u003e PM, LB, VK, KC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval: Yes,\u0026nbsp;\u003c/strong\u003eRef # IEC-AMS/AUH/75/2024-2025\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate:\u0026nbsp;\u003c/strong\u003eInformed written consent was obtained from each participants/parent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish:\u0026nbsp;\u003c/strong\u003eYes, informed written consent was obtained from each participant/parent.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBertelli, M.O., et al., \u003cem\u003eAutism spectrum disorder\u003c/em\u003e, in \u003cem\u003eTextbook of psychiatry for intellectual disability and autism spectrum disorder\u003c/em\u003e. 2022, Springer. p. 369-455.\u003c/li\u003e\n\u003cli\u003eShaw, K.A., \u003cem\u003ePrevalence and early identification of autism spectrum disorder among children aged 4 and 8 years\u0026mdash;Autism and Developmental Disabilities Monitoring Network, 16 Sites, United States, 2022.\u003c/em\u003e MMWR. 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Harding, \u003cem\u003eBrain renin-angiotensin\u0026mdash;a new look at an old system.\u003c/em\u003e Progress in neurobiology, 2011. \u003cstrong\u003e95\u003c/strong\u003e(1): p. 49-67.\u003c/li\u003e\n\u003cli\u003eMorales-Mar\u0026iacute;n, M.E., et al., \u003cem\u003eDifferential DNA methylation from autistic children enriches evidence for genes associated with ASD and new candidate genes.\u003c/em\u003e Brain Sciences, 2023. \u003cstrong\u003e13\u003c/strong\u003e(10): p. 1420.\u003c/li\u003e\n\u003cli\u003eStoccoro, A., et al., \u003cem\u003eDNA methylation biomarkers for young children with idiopathic autism spectrum disorder: a systematic review.\u003c/em\u003e International journal of molecular sciences, 2023. \u003cstrong\u003e24\u003c/strong\u003e(11): p. 9138.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Angiotensin II, AGTR2, Autism, Neurodevelopmental disorder","lastPublishedDoi":"10.21203/rs.3.rs-7040442/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7040442/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAutism Spectrum Disorder (ASD) is a complex neurodevelopmental condition with multifactorial unknown etiology that involves epigenetic influences. The epigenetic refers to the impact of environmental conditions on the nucleotide sequence. This exploratory study investigated the role of mutations in the \u003cem\u003eAGTR2\u003c/em\u003e gene, which encodes angiotensin II receptor type 2, in ASD pathogenesis. Six children with ASD participated in this study and their base pair nucleotide sequence was compared with the maternal relatives across three familial generations. Saliva samples were collected using swabbing technique, as the source for DNA extraction. Sanger sequencing was used to identify genetic mutations, especially the frameshift and point mutations in base pair nucleotide sequence of \u003cem\u003eAGTR2\u003c/em\u003e across three generations. Results revealed that the recurrent frameshift mutations in ASD affected individuals and their close relatives at 1-77 base pair length, suggesting potential heritable transmission. Point mutations were also observed although, their locations varied between individuals, indicating a possible contribution of de novo mutations. Consistently high adenine-thymine (A=T) content was found across all samples, suggesting genomic instability, which may act as a hotspot locus of mutation. Epigenetic mechanisms, such as DNA methylation and DNA Acetylation influenced by stressful environmental exposures were considered as the prime factor for the intergenerational inheritance of ASD susceptibility. These findings support the hypothesis that both inherited and environmental factors contribute to ASD risk, with \u003cem\u003eAGTR2\u003c/em\u003e emerging as a significant candidate gene for understanding complex etiology of ASD and supports the potential for \u003cem\u003eAGTR2\u003c/em\u003e related biomarkers in diagnosis and early intervention strategies.\u003c/p\u003e","manuscriptTitle":"Analysis of epigenetic mutation of AGTR2 gene responsible for Autism across three generation: An exploratory study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-28 05:29:52","doi":"10.21203/rs.3.rs-7040442/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3c6b65f2-c462-43a4-b2e8-0c52ff81bc22","owner":[],"postedDate":"July 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-04T13:11:42+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-28 05:29:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7040442","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7040442","identity":"rs-7040442","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}