Reduced cortical expression of a newly identified splicing variant of the DLG1 gene in patients with early-onset schizophrenia

Abstract

The human discs, large homolog 1 gene (DLG1) is mapped to the schizophrenia-susceptibility locus 3q29, and it encodes a scaffold protein that interacts with the N-methyl-D-aspartate receptor presumably dysregulated in schizophrenia. In the current study, we have newly identified a splicing variant of DLG1, which is transcribed from an unreported 95-base-pair exon (exon 3b) and is labeled 3b(+). We investigated the mRNA expression of 3b(+) in the post-mortem dorsolateral prefrontal cortices of patients with psychiatric disorders, obtained from The Stanley Medical Research Institute, and examined the potential association of the expression with the genotype of the single-nucleotide polymorphism (SNP) rs3915512 located within exon 3b. A real-time quantitative reverse transcriptase-polymerase chain reaction revealed that the mRNA levels of 3b(+) were significantly reduced in patients with early-onset schizophrenia (onset at <18 years old, P=0.0003) but not in those with non-early-onset schizophrenia, early-onset or non-early-onset bipolar disorder or in the controls. Furthermore, the genotype at the rs3915512 SNP was closely associated with the levels of 3b(+) mRNA expression. It is inferred that the T allele fails to meet the exonic splicing enhancer consensus, thus resulting in skipping of exon 3b, leading to the expression of 3b(-) (the previously known DLG1 variant) but not 3b(+). Because all the subjects with early-onset schizophrenia in the current study possess the T/T genotype, the reduced level of the DLG1 3b(+) transcript may be involved in the susceptibility and/or pathophysiology of early-onset schizophrenia.

Figure 1

A newly isolated human 3b(+) variant of DLG1 mRNA. (a) Detection of a novel splicing variant with a size of 314 bp, which contains 95 bp of exon 3b, above the major band with a size of 219 bp by reverse transcriptase-polymerase chain reaction (RT-PCR) from the human brain. The former PCR product was confirmed to be detected in different concentrations of complementary DNA (cDNA) templates: the cDNA used in the second lane was 10 times more dilute than that in the first lane. Control (−) indicates PCR without template DNA. Each number (base pairs) on the left indicates the migration position of the 100-bp DNA ladder. (b) Distribution of 3b(+) and 3b(−) transcripts in various human brain regions and peripheral organs. The total RNAs from each brain region and peripheral tissue were obtained from TakaraBio/Clontech. (c) Nucleotide sequence of the underlined exon 3b transcript and the deduced amino-acid residues of the predicted open reading frame in amino-acid symbols below the respective codons.

Figure 2

Structures of human DLG1 transcripts and proteins. (a) Schematic representation of the structure of human DLG1 transcript variants. The insertion of exon 3b between exons 3a (relabeled from original exon 3) and 4 might result in the premature termination of translation because of the in-frame stop codon in exon 3b (see b). *The new transcript variants with A and T alleles for rs3915512 were numbered AB855790 and AB855791, respectively. (b) Schematic representation of the structure of the putative proteins translated from the DLG1 transcript variants. The β- and α-form proteins are formed from the transcript variant 1–3 group and variant 4–5 group, respectively. From the transcript variant 6 including the 3b(+) message, a 65-amino-acid-long protein containing 15 unique C-terminal amino acids is formed. The arrowheads indicate the sites of the splicing variants.

Figure 3

3b(+) and 3b(−) mRNA expression in the dorsolateral prefrontal cortices (DLPFCs) for each diagnostic group. (a) Expression of 3b(+) mRNA. A significant decrease in 3b(+) mRNA expression was observed in the EOS group compared with the control subjects (P=0.0003, Welch's t-test). There were no significant differences between each of the other patient groups and control group. CT, control (n=34); EOS, early-onset schizophrenia (n=8); non-EOS, non-early-onset schizophrenia (n=26); EOBPs, early-onset bipolar disorders (n=7); non-EOBPs, non-early-onset bipolar disorders (n=26). (b) Expression of 3b(−) mRNA. Expression levels were normalized to that of GAPDH. Horizontal bars indicate means.

Figure 4

Outline of single-nucleotide polymorphism (SNP)-dependent insertion of exon 3b. (a) When the SNP rs3915512 consists of the T allele, the sequence (TGATAGAAT) does not meet the exonic splicing enhancer (ESE) consensus. Therefore, exon 3b skipping would tend to occur, leading to the production of the known DLG1 transcripts. (b) When the SNP consists of the A allele, the sequence (TGAAAGAAT) meets the ESE consensus, leading to the formation of an exon 3b-inserted splicing variant. The consensus of ESE was identified by Web analyses using ESEfinder (http://rulai.cshl.edu/tools/ESE/), FAS-ESS (http://genes.mit.edu/fas-ess/) and RESQUE-ESS (http://genes.mit.edu/burgelab/rescue-ese/).

Figure 5

Expression of 3b(+) variant transcript depending on the genotype of single-nucleotide polymorphism (SNP) rs3915512. (a) All samples from each diagnostic group are plotted together in two dimensions. Whereas subjects with genotypes containing the A allele (A/A or A/T) demonstrated a certain amount of 3b(+) mRNA expression, subjects with the T/T genotype demonstrated an extremely low expression. There were positive correlations between the 3b(+) and the 3b(−) mRNA expression for each genotype (A/A: n=5, r=0.97, P<0.01, A/T: n=31, r=0.88, P<0.001, T/T: n=65, r=0.85, P<0.001). The 3b(−) mRNA expression appears to be independent of genotype. Expression levels were normalized to that of GAPDH. (b) Genotypic frequencies in each diagnostic group. Relative frequencies of each genotype are shown for each diagnostic group. The numerical values in the rectangular bars are the number of subjects with the corresponding genotypes. The early-onset schizophrenia group included no subjects with genotypes containing the A allele (A/A or A/T), whereas some (29–42%) of subjects in the other groups had genotypes containing the A allele.