Association study of BDNF and DRD3 genes in schizophrenia diagnosis using matched case–control and family based study designs

Abstract

Schizophrenia (SCZ) is a severe neuropsychiatric disorder with prominent genetic etiologic factors. The dopamine receptor DRD3 gene is a strong candidate in genetic studies of SCZ because of the dopamine hypothesis of SCZ and the selective expression of D₃ in areas of the limbic system implicated in the disease. We examined 15 single-nucleotide polymorphisms (SNPs) in DRD3 in our sample of European origin consisting of 95 small nuclear SCZ families and 167 case–control pairs. We also examined four BDNF SNPs in our samples because of evidence for BDNF regulation of DRD3 expression (Guillin et al., 2001). We found a nominally significant genotypic association with rs7633291 and allelic association with rs1025398 alleles. However, these observations did not survive correction for multiple testing. We did not find a statistically significant association with the other DRD3 and BDNF polymorphisms. Taken together, the results from the present study suggest that BDNF and DRD3 may not be involved in SCZ susceptibility.

Introduction

Schizophrenia (SCZ) is a severe disabling neuropsychiatric disorder characterized by a collection of symptoms that may include positive symptoms such as paranoia and auditory hallucinations, negative symptoms including thought poverty, anhedonia, and social withdrawal, as well as cognitive impairment. It affects approximately 1% of the general population. Findings from family, twin, and adoption studies support a genetic basis for SCZ (reviewed in McGuffin et al., 1984), but its etiology is still unclear. Dopamine hypothesis of SCZ arose from the observations that dopamine-mimetic agents including amphetamine, induce SCZ associated symptoms, and that all antipsychotic medications have a certain degree of dopamine-blocking capacity.

D₃ is a G-protein coupled receptor (GPCR) that, upon binding of dopamine, transduces signal via inhibition of cyclic Adenosine Mono-Phosphate (cAMP) synthesis. D₃ is encoded by the DRD3 gene, which is located on chromosomal region 3q13.3. It is primarily expressed in the striatum, with the highest expression in the ventral portion. These brain regions have been implicated in SCZ pathophysiology. Ser9Gly is the only polymorphism that has been studied for its effects on D₃ function. The Gly allele has been associated with increased binding affinity of dopamine (Lundstrom and Turpin, 1996), as well as increased dopamine-induced stimulation of Extracellular signal Regulated Kinase (ERK), an important intracellular signaling molecule involved in cell growth and differentiation, and inhibition of cAMP synthesis (Jeanneteau et al., 2006).

Researchers found decreased DRD3 (Sokoloff et al., 1990) mRNA expression in postmortem brain samples (Schmauss et al., 1993) and peripheral blood lymphocytes (Vogel et al., 2004) of SCZ patients compared to those of controls. The decrease could be due to increased splicing of the 3′ region of the DRD3 pre-mRNA (Schmauss, 1996), leading to an increased ratio of truncated (D_3nf) to full-length (D₃) mRNA. In contrast, Ilani et al. (2001) found increased DRD3 mRNA levels in SCZ patients' blood lymphocytes compared to controls. The mixed results could be due to different housekeeping genes used (Ilani et al., 2001, Vogel et al., 2004). It is noteworthy that the influence of antipsychotic medication at the time of death could also have accounted for these discrepancies. In this regard, Gurevich et al. (1997) found increased D₃ levels in the ventral striatum of antipsychotic-free SCZ patients at the time of death, but similar D₃ levels in SCZ patients taking antipsychotics up to the time of death. These results suggest that while D₃ is up-regulated in SCZ, antipsychotics down-regulate it.

In the original genetic study of DRD3 in SCZ, Crocq et al. (1992) found Ser9Gly to be associated with SCZ using two matched case–control samples from France and the UK. Mant et al. (1994) from the same research group updated the positive findings with additional subjects, with an excess of the lower functioning Ser allele and Ser/Ser genotype in SCZ cases compared to controls. Our laboratory replicated the original positive findings using two independent samples from North America and Italy (Kennedy et al., 1995). The positive findings were also replicated in other independent samples (Shaikh et al., 1996, Spurlock et al., 1998, Ishiguro et al., 2000). However, they were challenged by results from many other studies, including the most recent meta-analyses (Ma et al., 2008, Utsunomiya et al., 2008).

Despite having accumulated genotype data from over 10,000 SCZ patients across different studies, with most studies having tested only the Ser9Gly polymorphism within this 50-kb gene, the role of DRD3 in SCZ remains elusive. Recently, Talkowski et al. (2006) investigated 11 polymorphisms evenly distributed within and around DRD3 for association with SCZ in two family samples (US and Indian) and a case–control sample. They found consistent association between Ser9Gly and SCZ in both family samples, but only against a common haplotype background (Talkowski et al., 2006). Dominguez and coworkers (2007) genotyped 17 polymorphisms spanning DRD3 and found haplotypes in the 3′ portion of the gene to be significantly associated in a Galician isolate population. These latest findings suggest that Ser9Gly may be a marker for other polymorphism(s) that confer risk to SCZ and encourage further more comprehensive examination of DRD3 in SCZ.

The brain-derived neurotrophic factor (BDNF) plays a critical role in dopaminergic neuronal establishment (Baquet et al., 2005). The number of tyrosine hydroxylase-expressing dopaminergic neurons was reduced in the midbrain–hindbrain regions where the BDNF gene was selectively deleted (Baquet et al., 2005). BDNF also specifically regulates the in-vivo expression of DRD3 in the nucleus accumbens both during development and in adulthood (Guillin et al., 2001). Postmortem studies in the brains of SCZ patients showed decreases in BDNF protein (Iritani et al., 2003, Weickert et al., 2003) and mRNA levels (Weickert et al., 2003), while Buckley et al., 2007, Palomino et al., 2006 recently found decreased plasma BDNF levels in first-episode patients. However, Takahashi et al. (2000) found increased BDNF protein levels in the hippocampus and nucleus accumbens in SCZ subjects. SCZ patients who were chronically on antipsychotic medications showed increased (Gama et al., 2007) or decreased (Grillo et al., 2007, Toyooka et al., 2002) serum BDNF levels compared to healthy controls. Similar BDNF levels between the chronically treated SCZ and controls were also reported (Shimizu et al., 2003). The mixed results could be due to a number of factors, including insufficient sample sizes, different patient populations, different brain regions examined, or different antipsychotics at different doses. Genetically, carriers of the BDNF Met66 allele have lower cognitive abilities of learning and memory associated with lower hippocampal activation compared to ValVal carriers (Egan et al., 2003). The mechanism of action could be reduced Met66-BDNF protein packaging and secretion from neurons compared to Val66-BDNF (Egan et al., 2003, Chen et al., 2004). The Met66 allele has also been associated with hippocampal volume (Pezawas et al., 2004). Five recent meta-analyses on Val66Met did not find a significant association with SCZ (Naoe et al., 2007, Qian et al., 2007, Xu et al., 2007a, Kanazawa et al., 2007, Zintzaras, 2007). The C270T polymorphism has also been analyzed in two meta-analyses, which found weak (Zintzaras, 2007) or no (Xu et al., 2007a) association with SCZ. However, the role of the BDNF gene could not be excluded as Qian et al. (2007) found a four-marker haplotype to be protective against SCZ in their Chinese sample. These results suggest that polymorphisms other than Val66Met and C207T may be involved in SCZ.

In the present study, we tested for the possibility of an association of tag polymorphisms in the DRD3 and BDNF genes with SCZ. We analyzed individual polymorphisms, as well as two-marker and three-marker haplotypes in a sliding window approach on our Caucasian paired case–control and small nuclear-family samples. In view of the substantive evidence that suggests a functional relationship between DRD3 and BDNF, we also examined their single-marker interactions for association with SCZ.