Establishing Methods For Screening Of Novel Sert Gene Polymorphisms

With an understanding of the layout of the SERT gene and an initial appreciation for critical regulatory regions, it is now possible to develop methods to efficiently evaluate the gene in human subjects for polymorphisms. We choose to bypass analysis of introns as this additional 35 kb of genomic DNA is largely unstudied and of unknown importance. Evaluating the known promoter region approximately doubles the analytical task, adding ~2 kb to the 1890 bp of exonic material that encompasses the SERT coding sequence. Considering whether to include regulatory regions in a polymorphism discovery effort is, of course, largely determined by the budget and scale of the screening effort. Because our lab is focused on the biology of transporter proteins, we are naturally most interested in variants that would affect the coding potential of SERT and have thus confined our initial analysis to exons and splice junctions.

Given that existing coding variants are rare, we prefer not to sequence genomic DNA but rather to adopt a pattern recognition approach that would allow one to determine whether a segment of DNA harbors sequence variation, as compared with a reference template. Our current method is an approach based on temperature-modulated, dHPLC (Giordano et al., 1999; Spiegelman et al., 2000) and is performed with a WAVE system from Transgenomics, Inc. (Omaha, NE, USA) This system allows for the evaluation of elution profiles of partially denatured, double-stranded DNAs in an automated, gel-free system. The technique is sensitive to single-base changes in DNA sequence in up to ~600 bp of amplified DNA. First, we establish PCR amplicons containing DNA of relatively uniform melting profiles (as determined by WAVEMAKER software; Transgenomics) and cover each exon with 20-60 bp of surrounding sequence to ensure incorporation of splice junctions. As intronic sequences often contain uninformative SNPs, we try to limit the amount of intronic sequence analyzed. In some cases, however, the placement of oligonuclotide primers is limited by the existing melting profiles of the genomic DNA. In some cases, we split our DNA segment into two amplicons, which allows a more uniform melting profile for each respective amplicon. For SERT, our PCR amplicons thus range from 200 to 500 bp and are generated on genomic DNA purified from 8-10 ml of whole blood (Puregene DNA Extraction Kit, Gentra Systems, Minneapolis, MN, USA). All blood collections and DNA analyses are performed by following informed consent of subjects under approved Institutional Review Board protocols. PCR reactions are performed on a Gene Amp PCR System 2400 by using AmpliTaq GoldTM DNA polymerase (Perkin Elmer-Applied Biosystems, Inc., Foster City, CA) and PfuTurbo DNA polymerase (Stratagene, Inc., La Jolla, CA) mixed at a ratio of 9:1. As the system is sensitive to single base errors, the inclusion of a polymerase with high 5 '-3' exonuclease activity is essential. Other polymerase mixtures can be used, although the columns deteriorate or can clog if used with unapproved buffer components; one is advised to consult closely with the manufacturer before branching out to new polymerase cocktails.

Before chromatography, a proband's SERT gene, in the form of PCR products (50 ng) for each exon, is mixed with the equivalent amplified exon from a presequenced, unaffected subject. In this manner, both heterozygous and homozygous mutations can be identified because both will contribute a proportion of mutant strands for annealing with control templates. Samples are denaturated at 95°C for 4 min, followed by gradual reannealing from 95°C to 25°C over 30 min and then injected into a DNASepR column with the mobile phase consisting of a linear acetonitrile gradient in 0.1 M triethylamine acetate buffer (TEAA), prepared by mixing of 0.1 M TEAA and 25% acetonitrile in 0.1 M TEAA. The calculated gradient, at a flow rate of 0.9 ml/min, is applied for all of the amplicons at column temperatures optimized for each amplicon. DNA is detected by UV absorbance, although one can implement a fluorescent detector if the oligonucleotides used for PCR amplification are end-labeled. Elution profiles for each amplicon are evaluated visually against chromatograms obtained from melted and re-annealed control samples to look for evidence of altered DNA elution, indicative of heteroduplex strand misalignment. All amplicons with suspicious elution profiles are sequenced on both strands by using amplicon or internal primers and fluorescent dideoxynucleotide terminators. This approach limits sequencing only to those templates suspected of harboring a sequence variant. Single-base mismatches are readily detected in this system. Figure 5.3 demonstrates the elution profile of an amplicon bearing a single base mutation compared with a control amplicon elution profile.

Next, the suspicious amplicon is sequenced by standard dideoxynucleotide approaches and, once the polymorphic base has been found, we perform a separate test to validate the presence of the polymorphism. If the sequence variation creates a unique restriction site, we digest amplicons from control and the proband and test for a restriction-fragment length polymorphism (RFLP). If an RFLP is not predicted, we utilize allele-specific

Figure 5.3. Identification of SERT polymorphisms using dHPLC. Shown are elution profiles for two amplicons of SLC6A4 following partial denaturation and chromatography on a WAVE dHPLC system. Note the readily distinguishable profiles of amplicons derived from a control SERT exon (bottom trace) and an amplicon bearing a heterozygous SNP (upper trace). Sequential processing of SERT amplicons on the WAVE system allows for efficient analysis of SERT sequence variations without recourse to DNA sequencing.

Figure 5.3. Identification of SERT polymorphisms using dHPLC. Shown are elution profiles for two amplicons of SLC6A4 following partial denaturation and chromatography on a WAVE dHPLC system. Note the readily distinguishable profiles of amplicons derived from a control SERT exon (bottom trace) and an amplicon bearing a heterozygous SNP (upper trace). Sequential processing of SERT amplicons on the WAVE system allows for efficient analysis of SERT sequence variations without recourse to DNA sequencing.

oligonucleotide (ASO) hybridization to validate the presence of the mutant allele (Shannon et al., 2000). In this latter strategy, subject and control amplicons are denatured onto nylon membranes and then hybridized to [32P]-labeled oligonucleotide probes carrying either the wild-type or the variant sequence. After the filters of excess probe have been washed, hybridization with the mutant oligonucleotide—but not the control oligonucleotide—is easily demonstrated on X-ray film.

As the dHPLC system is automated, samples can be loaded for unattended analysis. Our system has a 96-sample loader. Each run takes ~10 min, including time for column re-equilibration. If a gene has been divided into 15 amplicons, a single person's DNA can be evaluated in little more 2 h. Elution profiles are then evaluated for anomalous elution patterns and then either the amplicons are subjected to direct sequencing or another subject's DNA is analyzed. Our system allows for material for the analysis of approximately seven subjects to be loaded at a time. Once conditions for amplification have been set, this method provides a reasonably routine analysis of genomic structure with considerably lower sample costs than DNA sequencing. Pooling strategies, where multiple probands are combined for group analysis, can also be implemented to reduce costs further (Hoogendorn et al., 2000). The method is reasonably well suited to the sizes of samples available in clinical settings. For example, Glatt and co-workers (2001) reported SERT SNPs in 450 subjects drawn from the NHGRI Polymorphism Resource Panel (Collins et al., 1998). We have used this approach to evaluate juvenile obsessive-compulsive disorder (JOCD) subjects for novel SERT polymorphisms (Belous et al., 2000; Be-lous et al., manuscript in preparation) and are currently utilizing this method to explore SERT variation in major depressive disorder (MDD), irritable bowel syndrome (IBS), and IBS and MDD comorbid disorders (Belous et al., 2001). In our JOCD survey, we identified no novel coding variants but did detect a SNP (2631C) in exon 1b, the alternatively 5' noncoding spliced exon (Bradley and Blakely, 1997). Subsequent genotyping of this SNP in our JOCD subjects and controls reveals an increase in its frequency in affected subjects, which suggests that it may itself make a functional contribution to SERT mRNA expression or be linked to another informative variant.

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