Mutation screening of the DTNBP1 exonic sequence in 669 schizophrenics and 710 controls using high‐resolution melting analysis

Abstract

A large number of independent studies have reported evidence for association between the dysbindin gene (DTNBP1) and schizophrenia; however, specific risk alleles have been not been implicated as causal. In this study we set out to perform a comprehensive assessment of DNA variation within the exonic sequence of DTNBP1. To achieve this we optimized a high-resolution melting analysis (HRMA) protocol and applied it to screen all 11 DTNBP1 exons for DNA variants in a sample of 669 cases and 710 controls from the UK. Despite identifying seven exonic variants with a minor allele frequency (MAF) >0.01, none was significantly associated with schizophrenia (minimum P = 0.054), showing that the strong association we previously reported in this sample is not the result of association to a common functional variant located within the exonic sequence of any of the three major DTNBP1 transcripts. We also sought additional support for DTNBP1 as a susceptibility gene for schizophrenia by testing the hypothesis that rare exonic highly penetrant variants exist at the DTNBP1 locus. Our analysis failed to identify an enrichment of rare functional variants in the patients compared to the controls. Taken as a whole, this data demonstrate that if DTNBP1 is a risk gene for schizophrenia then risk is not conferred by mutations that affect the structure of the dysbindin protein.

Cell Biology of the BLOC-1 Complex Subunit Dysbindin, a Schizophrenia Susceptibility Gene

“…Based on the Ioannidis guidelines for the analysis of cumulative evidence in genetic association studies, the DTNBP1 association with disease is considered with a strong degree of epidemiologic reliability [24]. However, attempts to identify mutations in the exome of DTNBP1 in schizophrenia patients have been negative so far [25]. Moreover, the only human case reported carrying a loss-of-function allele in DTNBP1 lacked psychiatric manifestations.…”

Mutation screening of the 3q29 microdeletion syndrome candidate genes DLG1 and PAK2 in schizophrenia

“…HRMA was performed using a LightScannerÔ (Idaho Technologies) according to the manufacturer's instructions and as detailed in a recent study [Dwyer et al, 2010]: following PCR, each 12 ml sample was melted by increasing the temperature to 98 C at a rate of 0.1 C/sec with fluorescence data points being acquired continuously at a rate of 14 points/ C. The LightScanner platform includes Call-ITÔ software (Idaho Technologies). Call-ITÔ was used as follows: all melting curves were manually normalized by the user defining the temperature interval before and after the major change in fluorescence that correspond to 100% and 0% fluorescence, respectively.…”

“…HRMA analysis is performed in two-stages, where samples showing divergent profiles and dropouts undergo HRMA analysis again to confirm a divergent profile prior to sequencing. This protocol, followed by sequencing, allowed 100% identification of heterozygotes in a recent study by the same group [Dwyer et al, 2010].…”

High-resolution melting analysis of 15 genes in 60 patients with cytochrome-c oxidase deficiency

“…This approach is especially suitable for large-scale genetic studies. [20][21][22] Primers were designed, using the software Primer3Plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/ primer3plus.cgi), to amplify the coding regions of COX4I1, COX4I2, COX5A, COX5B, COX6A1, COX6A2, COX6B1, COX6C, COX7A1, COX7A2, COX7B, COX7C, COX8A, COX10 and COX15. Genomic DNA was amplified using PCR in the presence of LCGreen Plus Melting Dye (Idaho Technology Inc., Salt Lake City, UT, USA).…”