Functional Analysis of DNMT1 SNPs (rs2228611 and rs2114724) Associated with Schizophrenia

Sonal Saxena,G. Prasad Rao,Sumana Choudhury,Kiranmai Chennoju,Pranay Amruth Maroju,S. Ganesh Kumar,K. Naga Mohan,Poornima Kkani,Poonam Naik,Kumar Pranav Narayan,C. Ramasubramanian,Vidhya Chitta Voina,Josephina Meester,Trinath Jamma,Kavitha Gowdhaman

doi:10.1155/2021/6698979

Sonal Saxena, G. Prasad Rao

Open Access

https://doi.org/10.1155/2021/6698979

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Abstract

A recent study showed the association of minor alleles of rs2228611 (T allele) and rs2114724 (T allele) of DNMT1 with schizophrenia (SZ) and suggested their effects on splicing of the transcripts. We performed a replication study using 310 controls and 304 SZ patients and confirmed the association of the homozygous minor allele genotypes with SZ (P = 0.04 for rs2114724 and P = 0.007 for rs2228611). This significant association persisted after Bonferroni correction when the previously published data of 301 controls and 325 patients were also considered (P ≤ 0.0002). In addition, we found that the proportion of male patients with homozygous minor alleles at rs2114724 was significantly higher than that of females (P = 0.002). When haplotype analysis of both loci was performed, we observed a significant association of T/T–T/T and T/T–C/T (P = 0.04) haplotypes with SZ. To gain insights into the functional effects of the two SNPs on the levels of DNMT1 transcripts, quantitative real-time PCR experiments were performed using peripheral blood monocytes from 10 individuals each with T/T–T/T (homozygous minor allele), C/T–C/T (heterozygous), and C/C–C/C (homozygous major allele) haplotypes. Independently, the levels of DNMT1 protein were also compared in three individuals each by immunofluorescence. These results suggest that neither DNMT1 transcript nor the protein levels were significantly different in the peripheral blood monocytes among the individuals studied for the three groups. Taken together, our results confirm that the two minor alleles in homozygosity are associated with SZ but with no discernible effects on transcript or protein levels of DNMT1 in the peripheral blood monocytes of the small number of samples tested.

Highlights

Schizophrenia (SZ) is a complex disorder with a worldwide incidence of ∼0.7% and multiple etiologies that include genetic, environmental, and epigenetic mechanisms [1,2,3,4]
5–10 ml blood was drawn by venipuncture and the peripheral blood monocytes were isolated by using Histopaque 1077 (Merck, USA) solution. e purified monocytes were washed with 1X phosphate-buffered saline (1X PBS: 37 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4), used for isolation of genomic DNA using SDS-proteinase K method [12] and total RNA using RNeasy Mini kit (Qiagen, Germany)
The quantitative real-time PCR data revealed variation in the transcript levels within a haplotype, there was no difference in the average DNMT1 transcript levels between the three haplotypes (Figure 2(a))

Summary

Introduction

Schizophrenia (SZ) is a complex disorder with a worldwide incidence of ∼0.7% and multiple etiologies that include genetic, environmental, and epigenetic mechanisms [1,2,3,4]. Evidence for the involvement of epigenetic mechanisms in SZ comes from twin studies wherein monozygotic (MZ) twins show 41–65% concordance [1]. Of the different epigenetic processes in mammals, DNA methylation which involves addition of methyl group at the 5th carbon of cytosine is the most well studied [7]. DNA methylation is established by de novo methyltransferases DNMT3A and DNMT3B and maintained by DNMT1. DNMT3L has no catalytic activity but regulates de novo methylation [8]

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