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Keywords:

  • MIR137;
  • resequencing;
  • schizophrenia;
  • single nucleotide variation;
  • variable number of tandem repeats

Abstract

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

MicroRNA may play a role in the pathophysiology of schizophrenia. A recent meta-analysis of genome-wide association studies indicated a significant association between schizophrenia and a common intronic variation in MIR137HG (microRNA 137 host gene) encoding the primary microRNA-137. To explore additional risk variations for schizophrenia, we resequenced MIR137 and performed an association analysis in 1321 Japanese individuals. By resequencing, we detected four sequence variations in the 5' and 3' flanking regions. There were no significant associations between these variations and schizophrenia. Our resequencing and association analysis of MIR137 failed to find additional risk variations for schizophrenia.

MICRORNA (MIRNA), SMALL non-coding RNA, are key modulators of post-transcriptional gene regulation and have a role in synaptogenesis and neuronal plasticity.[1] Dysregulation of miRNA may be a factor in the neurobiological mechanisms underlying schizophrenia.[2]

Neurons are enriched with miRNA-137, an miRNA that regulates neuronal maturation.[3] A recent meta-analysis of genome-wide association studies (GWAS) indicated a significant association between schizophrenia and a common intronic single nucleotide variation (SNV) in MIR137HG (the microRNA 137 host gene), which encodes the primary miRNA-137.[4] To further explore risk variations for schizophrenia, we resequenced MIR137 and performed an association analysis in 1321 Japanese individuals.

Methods

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The present study was approved by the Ethics Committee of Genetics at the Niigata University School of Medicine, and written informed consent was obtained from all participants. All participants were unrelated and of Japanese descent. The study population comprised 647 patients (349 men and 298 women; mean age 39.8 ± 13.8 years) with schizophrenia diagnosed according to the DSM-IV criteria and 674 mentally healthy individuals (341 men and 333 women; mean age 38.4 ± 10.8 years), with no personal or family history (within first-degree relatives) of psychiatric disorders. A psychiatric assessment of each participant was conducted, as previously described.[5]

MIR137 was resequenced in 1321 individuals using direct sequencing of polymerase chain reaction (PCR) products as previously described.[6] Forward and reverse primer sequences for amplification were 5'-GATTTATGGTCCCGGTCAAG-3' and 5'-CTTTCCGGTGGAACCAGTG-3', respectively. We analyzed a variable number of tandem repeats (VNTR) detected by resequencing using fragment analysis of PCR products with fluorescently labeled forward and unlabeled reverse primers using the 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) with GeneMapper v3.7 software (Applied Biosystems, Foster City, CA, USA).

Deviations from the Hardy–Weinberg equilibrium (HWE) of the SNV and the VNTR were tested using Haploview v4.2 (http://www.broadinstitute.org/scientific-community/science/programs/medical-and-population-genetics/haploview/haploview) and the GENEPOP v4.0.10 program (http://genepop.curtin.edu.au/), respectively. We compared overall allelic distributions of the VNTR between patients and control groups using CLUMP v2.4 (http://www.smd.qmul.ac.uk/statgen/dcurtis/software.html). We examined each individual allele of the SNV and the VNTR for an association with schizophrenia using Fisher's exact test. A power calculation was performed using the Genetic Power Calculator (http://pngu.mgh.harvard.edu/~purcell/gpc/). The power was estimated with an α of 0.05, assuming that the disease prevalence was 0.01 and the risk allele frequencies of SNV to be the value observed in controls.

Results

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

By resequencing, we detected four sequence variations (three SNV and one VNTR) in the 5' and 3' flanking regions, but not in the gene region. Of these, two had previously been reported: SNV1 (rs150014880) in the 3' flanking region and a VNTR comprising two to nine repeats of a 15 bp sequence starting in the flanking region 6 bp from the 5' end of MIR137.[7] The other two SNV, SNV2 (g.98511769G > T) and SNV3 (g.98511780T > C) in the 5' flanking region, were previously unidentified. Three SNV were not associated with schizophrenia (Table 1). The genotype distributions of the VNTR significantly deviated from the HWE only in the schizophrenia group (P = 0.002). There were no significant associations between the VNTR and schizophrenia (Table 2).

Table 1. Allelic association analysis of the SNV with schizophrenia
SNV#VariationaSchizophreniaControlP
1b2b1b2b
  1. a

    Position on Homo sapiens chromosome 1, GRCh37.p10 Primary Assembly (NC_000001.10).

  2. b

    Ancestral and mutant alleles are denoted by 1 and 2, respectively.

  3. SNV, single nucleotide variation.

SNV1g.98511534G > C12940134711.000
SNV2g.98511769G > T12868134350.414
SNV3g.98511780T > C12940134711.000
Table 2. Allelic association analysis of the VNTR with schizophrenia
AlleleSchizophreniaControlPa
  1. a

    Global P = 0.315. The number of simulations was 10 000, and the T1 statistic was adopted.

  2. VNTR, variable number of tandem repeats.

2111.000
39159790.280
42552530.554
573550.070
627230.480
715260.118
88100.815
9011.000

Discussion

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

In the present study, we resequenced MIR137 in 1321 Japanese individuals and detected four sequence variations (three SNV and one VNTR) in the 5' and 3' flanking regions. Among these variations, the VNTR is suspected of being functional. In melanoma cell lines, a 12 repeat showed less effective processing of MIR137 and translational suppression of an MIR137 target than a 3 repeat.[7] We failed to find associations between schizophrenia and the four variations, including the functional VNTR.

Recently, Xu et al. identified nonsense and missense de novo mutations of DPYD (dihydropyrimidine dehydrogenase) at 1q21.3 in two unrelated probands with schizophrenia by exome sequencing in 231 trios (probands and both their parents), whereas they did not find mutations of MIR137.[8] We also did not detect sequence variations in the MIR137 gene region. In the most recent schizophrenia GWAS meta-analysis, a 1q21.3 locus containing DPYD and MIR137 showed a nearly genome-wide significant association.[9] This association may reflect the contribution of DPYD to schizophrenia.

A major limitation of the current study is that the sample size of our population may not have sufficient statistical power to detect associations between schizophrenia and rare SNV identified by resequencing. A power analysis showed that the power was 0.20–0.71, assuming a genotypic relative risk of 4.0 for heterozygous risk allele carriers under the multiplicative model of inheritance. Thus, we could not exclude the possibility that our negative results were caused by type II errors.

In conclusion, our resequencing and association analysis of MIR137 failed to find further risk variations for schizophrenia.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

This work was supported by a Grant-in Aid for Scientific Research (#23791312) and a Grant for Promotion of Niigata University Research Projects (#24C031). There is no conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References