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Keywords:

  • association study;
  • autism;
  • chromosome 15;
  • ryanodine receptor;
  • single nucleotide polymorphism

Abstract

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Aim:  Autism is a neurodevelopmental disorder with a complex genetic etiology. Chromosome 15q11-q14 has been proposed to harbor a gene for autism susceptibility because deletion of the region leads to Prader-Willi syndrome or Angelman syndrome, having phenotypic overlap with autism. Here we studied the association between autism and the ryanodine receptor 3 (RyR3) gene, which is located in the region. This is the first study, to our knowledge, that has investigated the association.

Methods:  We genotyped 14 tag single nucleotide polymorphisms (SNPs) in 166 Japanese patients with autism and 375 controls.

Results:  No significant difference was observed between the patients and controls in allelic frequencies or genotypic distributions of the 14 SNPs. Analysis after confining the subjects to males showed similar results.

Conclusions:  The present study provides no positive evidence for the association between the RyR3 gene and autism in the Japanese population.

AUTISM IS A developmental disorder characterized by three areas of abnormality: impairment in social interaction, impairment in communication, and restricted and stereotyped pattern of interest or behavior. Impairment in all three areas is observed before the age of 3 years and disrupted growth of the brain, with unknown mechanism, is implicated in the etiology of autism. Twin and family studies have indicated a robust role of genetic factors in the development of autism, while few susceptibility genes have been elucidated.1 Chromosome 15q11-q14 is a region highly susceptible to clinically important genomic rearrangements, including interstitial deletions, duplications, triplications, and the generation of supernumerary marker chromosomes (SMCs), called ‘idic’ or ‘inverted duplication’.2 Deletions of the region lead to Prader-Willi syndrome and Angelman syndrome depending on the deleted chromosome's parent of origin.3 Both syndromes have phenotypic overlap with autism therefore chromosome 15q11-q14 has been proposed to harbor a gene for autism susceptibility.4,5

The ryanodine receptor 3 (RyR3) gene has been mapped to chromosome 15q14-q15.6 It is an isoform of RyRs and expressed at high levels in caudate nucleus, amygdala, and hippocampus in the central nervous system.7 An animal study showed that deletion of the RyR3 changes hippocampal synaptic plasticity, specifically on the adaptation of acquired memory in response to external changes or stimuli, without affecting hippocampal morphology, basal synaptic transmission or presynaptic function.8 Calcium-induced calcium release from a ryanodine-sensitive Ca2+ store may be required for the induction of contextual learning.9 Restricted and stereotyped pattern of interest or behavior without adjusting external changes or stimuli is one of the three characteristics of autism. On the basis of the location and function of the gene, RyR3 may be a candidate for autism susceptibility. To our knowledge, however, no study has investigated the genetic association between RyR3 and autism to date. Here we investigate the association between the RyR3 gene and autism in a Japanese population.

METHODS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Subjects

In this study, Japanese patients and control subjects around Tokyo, Japan, were recruited: 166 unrelated patients with autistic disorder (147 males and 19 females; age, 19.9 ± 9.8 years, mean ± SD) and 375 unrelated healthy volunteers (127 males and 248 females; age: 36.0 ± 11.7 years). Two experienced child psychiatrists independently conducted a semistructured behavior observation of the subjects, interviewed all patients and their parents, and made final diagnoses according to the ICD-10 DCR and DSM-IV. The cases that fulfilled both ICD-10 and DSM-IV criteria were included in the present study and were followed up for 6 months. We excluded the cases that were found not to fulfill both criteria within the period. At the interview, the Child Behavior Questionnaire Revised10 was used to assist the evaluation of the autism-specific behaviors and symptoms. In order to exclude other genetic syndromes, we performed standard karyotyping and fragile X-testing for the trinucleotide repeat expansion in the FMR-1 gene.11 IQ levels were >70 in 12 patients, 50–70 in 33 patients, 35–50 in 30 patients, and <35 in 37 patients. The levels were evaluated mainly using a Japanese version of the Binet test. Thirty-nine patients were unable to take the IQ test due to their communication disorders or disability to understand the questions. Data were not available on the other 15 patients. The objective of the present study was clearly explained, and written informed consent was obtained from all parents. Consent was also obtained from the patients when they were able to follow the explanation (or when mental age was approximately >6 years old). Controls were mainly recruited from the hospital and facility staff. All controls had a short interview with one of the authors to confirm that they had no history of major mental illness. The study was approved by the Ethical Committee of the Faculty of Medicine, the University of Tokyo.

Genotyping

Genomic DNA was extracted from leukocytes using the standard phenol-chloroform method. We genotyped 14 single nucleotide polymorphisms (SNPs) as detailed in Table 1. All regions of the gene were covered by selecting tag SNPs by using SNP Wizard module (SNP Tag Selection, pair-wise r2 = 99%) of SNPbrowserTM software (Applied Biosystems, Foster City, CA, USA), in which haplotype blocks are estimated depending on the data from the International HapMap project12 or Applied Biosystems. All SNPs were analyzed by TaqMan PCR method using the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems).

Table 1.  Allele and genotype frequencies of 14 single nucleotide polymorphisms in the RYR3 gene
SNPsdb SNP IDLocationAlleles (Major/Minor)Minor allele frequencyGenotype frequencies**Chromosome position (bp)
Autism*Control*AutismControl
  • *

    Number of genotyped individuals for each SNP is given in parenthesis.

  • **

    Described as major homo/hetero/minor homo.

  • SNP, single nucleotide polymorphisms.

SNP1rs2676052Intron 1C/T0.37 (166)0.35 (365)0.38/0.50/0.120.43/0.45/0.1210483443
SNP2rs2596175Intron 2T/A0.44 (166)0.46 (371)0.31/0.50/0.190.29/0.50/0.2110581450
SNP3rs1435118Intron 12A/G0.39 (166)0.44 (370)0.39/0.45/0.160.31/0.50/0.1910667021
SNP4rs640152Intron 16G/A0.27 (166)0.26 (372)0.55/0.35/0.100.55/0.39/0.0610686057
SNP5rs2467565Intron 16A/G0.26 (166)0.21 (371)0.55/0.39/0.060.61/0.35/0.0410694156
SNP6rs1495280Intron 25T/C0.35 (166)0.33 (368)0.43/0.44/0.130.44/0.45/0.1110729162
SNP7rs937303Intron 30A/G0.40 (166)0.39 (375)0.36/0.48/0.160.37/0.47/0.1610742164
SNP8rs12907278Intron 38G/A0.48 (166)0.43 (374)0.30/0.45/0.250.31/0.51/0.1810774578
SNP9rs2293027Exon 44G/A0.20 (166)0.22 (371)0.63/0.35/0.020.61/0.34/0.0510816402
SNP10rs2288606Intron 49C/A0.27 (166)0.27 (373)0.54/0.37/0.090.53/0.39/0.0810829532
SNP11rs12916967Intron 62A/G0.44 (164)0.43 (373)0.32/0.48/0.200.31/0.53/0.1610873068
SNP12rs11072673Intron 67G/T0.42 (164)0.45 (372)0.32/0.52/0.160.31/0.47/0.2210897620
SNP13rs1036005Intron 75T/C0.25 (164)0.25 (373)0.55/0.41/0.040.59/0.33/0.0810914237
SNP14rs713202Intron 95C/T0.26 (165)0.31 (349)0.53/0.42/0.050.48/0.43/0.0910949706

Statistical method

The chi-square test was used to compare the allele or genotype frequencies between the patients and controls. Lewontin's D′ was used to analyze pairwise linkage disequilibrium (LD).13 Haplotype block analysis was conducted in the Gabriel method as well as the Four Gamete method.14,15 Haploview 3.3216 was used to conduct LD and haplotype block analyses. Statistical power was calculated by using the Genetic Power Calculator.17

RESULTS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Table 1 shows the allele and genotype frequencies of the 14 SNPs compared between the patients and controls. The distributions of all 14 SNPs follow the Hardy-Weinberg equilibrium both in the patients and controls. No significant difference was observed in allelic frequencies or genotypic distributions of the 14 SNPs between the patients and controls. Analysis after confining the subjects to males showed no significant difference (data not shown).

The strength of LD denoted as D′ between pairs of SNPs is shown in Fig. 1. No haplotype block was suggested by the Gabriel or Four Gamete method of haplotype block analysis,14,15 which had been expected considering the definition of tag SNPs. The LD analysis in males showed similar results (data not shown).

image

Figure 1. The strength of linkage disequilibrium between pairs of single nucleotide polymorphisms in all subjects. The strength of linkage disequilibrium is denoted as D′ (100 × exact D′ value).

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DISCUSSION

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

In the present study, we investigated the possible association between the RyR3 gene and autism by analyzing 14 tag SNPs. No significant difference was observed between the patients and controls in allelic frequencies or genotypic distributions of the 14 SNPs. Analysis after confining the subjects to males showed similar results. Thus, the present study provides no positive evidence for the association between the RyR3 gene and autism in the Japanese population.

Statistical power of the present study is 0.76 (α = 0.05) when assuming that the prevalence of autism is 0.21% in the Japanese population,18 genotypic relative risk is 1.8 (dominant model), and the risk allele frequency is 0.1. Thus, our results might have adequate statistical power to detect the effect of the gene with Odds ratios of approximately 1.8 or more at nominal P-value of 0.05, although the power is reduced when multiple testing is considered and also smaller effects might not be detected in the present sample. Caution may be needed for the controls in the present study because they were not age-matched to the patients. However, this may not be likely to significantly affect the result, considering no major effect of environmental factors in autism.19 An imbalance in sex ratio between the patients and controls may be overcome by analysis confining the subjects to males considering its higher prevalence in males than in females. A wide range of IQ levels in the patients may be another limitation in the present study. Heterogeneity in the present subjects might affect the results.

In conclusion, no evidence was obtained for a role of the RyR3 gene in the development of autism. However, rare variant or epigenetic factors were not investigated in the present study. Further investigation of the region including these factors may be recommended.

REFERENCES

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES