Resequencing and association analysis of OXTR with autism spectrum disorder in a Japanese population

Authors

  • Jun Egawa MD, PhD,

    1. Department of Psychiatry, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
    2. Department of Pediatric Psychiatry, Center for Transdisciplinary Research, Niigata, Japan
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  • Yuichiro Watanabe MD, PhD,

    Corresponding author
    1. Department of Psychiatry, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
    2. Division of Medical Education, Comprehensive Medical Education Center, School of Medicine, Faculty of Medicine, Niigata, Japan
    • Correspondence: Yuichiro Watanabe, MD, PhD, Department of Psychiatry, Niigata University Graduate School of Medical and Dental Sciences, 757 Asahimachidori-ichibancho, Chuo-ku, Niigata 951-8510, Japan. Email: yuichiro@med.niigata-u.ac.jp

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  • Masako Shibuya MD, PhD,

    1. Department of Psychiatry, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
    2. Health Administration Center, Headquarters for Health Administration, Niigata University, Niigata, Japan
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  • Taro Endo MD, PhD,

    1. Department of Psychiatry, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
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  • Atsunori Sugimoto MD,

    1. Department of Psychiatry, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
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  • Hirofumi Igeta MD,

    1. Department of Psychiatry, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
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  • Ayako Nunokawa MD, PhD,

    1. Department of Psychiatry, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
    2. Oojima Hospital, Niigata, Japan
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  • Emiko Inoue MD,

    1. Department of Psychiatry, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
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  • Toshiyuki Someya MD, PhD

    1. Department of Psychiatry, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
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Abstract

Aims

The oxytocin receptor (OXTR) is implicated in the pathophysiology of autism spectrum disorder (ASD). A recent study found a rare non-synonymous OXTR gene variation, rs35062132 (R376G), associated with ASD in a Japanese population. In order to investigate the association between rare non-synonymous OXTR variations and ASD, we resequenced OXTR and performed association analysis with ASD in a Japanese population.

Methods

We resequenced the OXTR coding region in 213 ASD patients. Rare non-synonymous OXTR variations detected by resequencing were genotyped in 213 patients and 667 controls.

Results

We detected three rare non-synonymous variations: rs35062132 (R376G/C), rs151257822 (G334D), and g.8809426G>T (R150S). However, there was no significant association between these rare non-synonymous variations and ASD.

Conclusions

Our present study does not support the contribution of rare non-synonymous OXTR variations to ASD susceptibility in the Japanese population.

Autism spectrum disorder (ASD) is marked by social and communication deficits, and the presence of rigid and repetitive behaviors and interests. It is a complex disorder with a heritability of 0.8.[1]

The peptide hormone oxytocin (OXT) has a role in social functions, such as emotion recognition and feelings of attachment.[2] OXT actions are mediated through the OXT receptor (OXTR), which belongs to the class I G-protein coupled receptor family.[3] OXT-OXTR signaling has been implicated in ASD pathophysiology.[4] Thus, the OXTR gene is a potential candidate gene for ASD.

Several studies have found common non-coding OXTR variations associated with ASD,[5-10] although a recent meta-analysis did not find any association between ASD and two of the most extensively investigated variations, rs53576 and rs2254298, located in intron 3.[11] Almost all earlier studies investigated common, and not rare, variations. Recently, Ma et al.[12] demonstrated that a rare non-synonymous variation, rs35062132 (R376G), detected by resequencing of the OXTR coding region in 59 ASD patients and 30 control individuals, was associated with ASD in a Japanese population (132 patients and 248 controls). To confirm the association between rare non-synonymous OXTR variations and ASD, we resequenced the OXTR coding region in 213 ASD patients, and then performed a case–control study (213 patients and 667 controls).

Methods

The study was approved by the Ethics Committee on Genetics of the Niigata University School of Medicine, and written informed consent was obtained from all participants and/or their families. All participants were unrelated and of Japanese descent. The study population consisted of 213 patients with ASD (168 were male and 45 were female; mean age, 17.2 [SD 8.2] years) and 667 control individuals (341 were male and 326 were female; mean age, 38.3 [SD 10.8] years). Patient and control groups were not sex- or age-matched. Each participant was subjected to psychiatric assessment, as previously described.[13, 14] In brief, patients were diagnosed according to the DSM-IV criteria for autistic disorder (n = 66), Asperger's disorder (n = 99), or pervasive developmental disorder not otherwise specified (NOS; n = 48). Diagnoses were made by experienced child psychiatrists, and based on all available information, including unstructured interviews of patients and their families, clinical observation, and examination of medical records, although we did not use standardized tests, such as the Autism Diagnostic Interview-Revised or the Autism Diagnostic Observation Schedule. We ruled out syndromic ASD, for example, fragile X syndrome, tuberous sclerosis, and Rett syndrome. ASD patients had co-morbidities, including mental retardation (n = 40), epilepsy (n = 5), obsessive–compulsive disorder (n = 5), selective mutism (n = 1), trichotillomania (n = 1), schizophrenia (n = 1), psychotic disorder NOS (n = 1), and depressive disorder NOS (n = 1). Controls were mainly recruited from hospital staff, and showed good social and occupational skills with no self-reported personal or family history (within first-degree relatives) of psychiatric disorders, but were not assessed using structured psychiatric interviews.

The OXTR coding region was resequenced in 213 ASD patients by direct sequencing of polymerase chain reaction products, as previously described.[15] Primer sequences used for amplification are listed in Table S1. Detailed information on amplification conditions is available upon request. Rare non-synonymous variations with minor allele frequencies < 0.05 were genotyped in the study population using the TaqMan 5′-exonuclease assay (Applied Biosystems, Foster City, CA, USA), as previously described.[14]

Deviations from Hardy–Weinberg equilibrium (HWE) were tested using Haploview v4.2 (http://www.broadinstitute.org/scientific-community/science/programs/medical-and-population-genetics/haploview/haploview) or genepop v4.0.10 (http://genepop.curtin.edu.au/). Allelic associations were tested using Fisher's exact test. A power calculation was performed using the Genetic Power Calculator (http://pngu.mgh.harvard.edu/~purcell/gpc/). Power was estimated using α = 0.05, and assuming a disease prevalence of 0.009.[1]

Results

Resequencing the OXTR coding region, we detected eight coding sequence variations in 213 ASD patients (Fig. 1; Table S2). Of these, three were rare non-synonymous variations: rs35062132 (g.8794707G>C; R376G), rs151257822 (g.8794832C>T; G334D) and g.8809426G>T (R150S). Thus, we genotyped rs35062132 (R376G/C), rs35062132 (R376C), and g.8809426G>T (R150S) in 213 patients and 668 controls (Table 1). In all 213 patients, genotypes of these variations determined using the TaqMan method were identical to those obtained by direct sequencing. Genotype distributions of the variations did not deviate significantly from HWE in the ASD and control groups (data not shown). None of the allele frequencies of the variations differed significantly between the two groups. In addition, analyzing male and female subjects separately, we found no significant associations between the variations and ASD (data not shown).

Figure 1.

Genomic structure of the oxytocin receptor (OXTR) gene. OXTR has four exons (rectangles) and spans approximately 19.2 kb. Shaded and white rectangles represent coding and untranslated regions, respectively. A horizontal arrow shows the transcriptional orientation. Vertical arrows indicate locations of coding sequence variations identified by resequencing.

Table 1. Association analysis between rare non-synonymous OXTR variations and ASD
VariationTaqMan SNP Assay IDGenotypeMAFAllelic p
  1. aPrimer and reporter sequences; forward primer: 5′-AGCAACTCGTCCTCCTTTGTC-3′; reverse primer: 5′-GAGCAGCTCCTCTGGCT-3′; reporter 1: 5′-VIC-AGCTGCGATGGCTC-NFQ-3′; reporter 2: 5′-FAM-AGCTGCAATGGCTC-NFQ-3′.
  2. bPrimer and reporter sequences; forward primer: 5′-GAAGCGCTGCACGAGTTC-3′; reverse primer: 5′-CTGCAACCCCTGGATCTACATG-3′; reporter 1: 5′-VIC-CTGTTCACGGGCCACCT-NFQ-3′; reporter 2: 5′-FAM-CTGTTCACGGACCACCT-NFQ-3′.
  3. cPrimer and reporter sequences; forward primer: 5′-CGCTGCCTGGCCATCT-3′; reverse primer: 5′-CGTGGCGAGCACTGC-3′; reporter 1: 5′-VIC-CTGCGCCGCCGCA-NFQ-3′; reporter 2: 5′-FAM-CTGCGCAGCCGCA-NFQ-3′.
  4. ASD, autism spectrum disorder; MAF, minor allele frequency; OXTR, oxytocin receptor.
rs35062132 (R376G/C)C__62467197_10/ CustomaG/GG/CG/AC/CA/AGAGA
ASD 20940000.00900.801
Control 649162000.0120.002  
rs151257822 (G334D)CustombC/CC/TT/T  T T 
ASD 21210  0.002 0.69 
Control 66070  0.005   
g.8809426G>T (R150S)CustomcG/GG/TT/T  T T 
ASD 21210  0.002 0.43 
Control 66610  0.001   

Discussion

We resequenced the OXTR coding region in 213 ASD patients, and detected three rare non-synonymous variations: rs35062132 (R376G), rs151257822 (G334D), and g.8809426G>T (R150S). Of note, rs35062132 (R376G/C) may be functional. OXT-induced receptor internalization and recycling are faster in human embryonic kidney 293 (HEK-293) cells expressing OXTR-376G, than in those expressing OXTR-376R or OXTR-376C.[12] Additionally, agonist-induced elevation of intracellular free calcium concentrations is smaller in both HEK-293 and neuroblastoma/glioma hybrid cells expressing OXTR-376G or OXTR-376C tagged with enhanced green fluorescent protein (EGFP), than those expressing OXTR-376R tagged with EGFP.[12] While the functional implications of rs151257822 (G334D) and g.8809426G>T (R150S) are still unknown, possible effects of these rare non-synonymous variations on OXTR function are predicted as ‘probably damaging’ by PolyPhen-2 v2.2.2 (http://genetics.bwh.harvard.edu/pph2/). Functional analysis is required to assess the accuracy of this prediction.

In our Japanese population (213 patients and 667 controls), there was no significant association between ASD and any of the rare non-synonymous variations. Ma et al.[12] found that the 376G allele of rs35062132 is associated with ASD in a Japanese population (132 patients and 248 controls). However, there are differences in the 376G allele frequency in controls, between this study and our own (0.004 and 0.012, respectively; Table S3). In our 667 controls, the 376G allele frequency (0.012) is similar to that reported (0.010) in 1148 Japanese individuals from the Human Genetic Variation Database (http://www.genome.med.kyoto-u.ac.jp/SnpDB/). Genotypes determined using TaqMan and direct sequencing were identical, therefore the likelihood of genotyping errors appears low. The inconsistency between the studies may be due to the insufficient statistical power of individual studies. The sample size of our population (n = 880) was larger than that of Ma et al.[12] (n = 380), but still may not provide adequate power to detect associations between rare variations and ASD. The statistical power of our sample size was 0.36, assuming a risk allele frequency of 0.01 and a genotypic relative risk of 2.0 for heterozygous risk allele carriers under the multiplicative model of inheritance. Thus, we cannot exclude the possibility that the negative results we obtained are caused by type II errors. When we combined samples from both studies, the results were still negative (Table S4). The genetic effect size of rs35062132 may be overestimated in the study by Ma et al.[12] To draw a definitive conclusion, further studies should be performed using sufficiently large sample sizes.

We recognize the limitations of our present study. First, the patient and control groups were not age- or sex-matched, although this was also the case in the study by Ma et al.[12] Fein et al.[16] demonstrated that there are individuals with a history of ASD but who no longer show any significant autistic impairments. Therefore, we cannot exclude the possibility that our controls may include some individuals with early histories of ASD. Prevalence of ASD is higher in boys than girls,[1] therefore we analyzed male and female subjects separately, but did not find any significant associations. Second, our participants were not assessed using standardized structured interviews, and the controls were not well characterized. Accordingly, there is the possibility that our failure to find an association may be due to misdiagnosis.

In conclusion, our resequencing and association analysis of OXTR does not provide supportive evidence for the contribution of rare non-synonymous OXTR variations to ASD susceptibility in the Japanese population.

Acknowledgments

The authors thank the patients, their families, and the healthy volunteers for participation; Drs Sugiyama, Masuzawa, Kaneko, and Tamura for their help in recruiting participants; Ms Yamazaki for excellent technical assistance; and Drs Algovik, Iida, Kim, and Yokoyama for kindly providing resequencing information. None of the authors have any conflicts of interest to declare.

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