Characterisation of Genetic Variation in <i>ST8SIA2</i> and Its Interaction Region in NCAM1 in Patients with Bipolar Disorder

<div><p>Alpha-2,8-sialyltransferase 2 (ST8SIA2) is an enzyme responsible for the transfer of polysialic acid (PSA) to glycoproteins, principally the neuronal cell adhesion molecule (NCAM1), and is involved in neuronal plasticity. Variants within <i>ST8SIA2</i> have previously shown association with bipolar disorder, schizophrenia and autism. In addition, altered PSA-NCAM expression in brains of patients with schizophrenia or bipolar disorder indicates a functional dysregulation of glycosylation in mental illness. To explore the role of sequence variation affecting PSA-NCAM formation, we conducted a targeted re-sequencing study of a ∼100 kb region – including the entire ST8SIA2 gene and its region of interaction with NCAM1 – in 48 Caucasian cases with bipolar disorder using the Roche 454 platform. We identified over 400 DNA variants, including 47 putative novel variants not described in dbSNP. Validation of a subset of variants via Sequenom showed high reliability of Roche 454 genotype calls (97% genotype concordance, with 80% of novel variants independently verified). We did not observe major loss-of-function mutations that would affect PSA-NCAM formation, either by ablating ST8SIA2 function or by affecting the ability of NCAM1 to be glycosylated. However, we identified 13 SNPs in the UTRs of <i>ST8SIA2</i>, a synonymous coding SNP in exon 5 (rs2305561, P207P) and many additional non-coding variants that may influence splicing or regulation of <i>ST8SIA2</i> expression. We calculated nucleotide diversity within <i>ST8SIA2</i> on specific haplotypes, finding that the diversity on the specific “risk” and “protective” haplotypes was lower than other non-disease-associated haplotypes, suggesting that putative functional variation may have arisen on a spectrum of haplotypes. We have identified common and novel variants (rs11074064, rs722645, 15∶92961050) that exist on a spectrum of haplotypes, yet are plausible candidates for conferring the effect of risk and protective haplotypes via multiple enhancer elements. A Galaxy workflow/pipeline for sequence analysis used herein is available at: <a href="https://main.g2.bx.psu.edu/u/a-shaw-neura/p/next-generation-resources" target="_blank">https://main.g2.bx.psu.edu/u/a-shaw-neura/p/next-generation-resources</a>.</p></div

SNPs nominally associated with bipolar disorder.

<p>The single nucleotide polymorphisms (SNP) nominally associated with bipolar disorder (p<0.05) are given. The SNP position on chromosome 15 is given relative to hg19 build. The reference (Ref) and alternative (Alt) alleles are shown, with the frequency (Freq) of the alternative allele in affected individuals (n = 210 cases) and unaffected controls (n = 160) shown. Odds ratio of effect and the 95% confidence interval (CI) are given.</p

Genotype concordance between 454-generated genotype calls and those directly genotyped via Illumina GoldenGate or Illumina 660 W.

<p>Genotype calls are divided into two categories: all calls; or confident genotype calls based on Genotype Quality (GQ) ≥ 10. Call accuracy gives the concordance between the SNP genotype derived from 454 data and Illumina-generated data across all samples excluding missing data, and the call rate incorporates missing data on an individual basis. These are calculated for all genotypes, and heterozygous (het) calls only.</p

Haplotype phasing of all detected nucleotide variants in <i>ST8SIA2</i> gene region.

<p>The gene structure is shown above, with the minor allele frequency of each SNP detected shown by the histogram immediately below the gene (red represents frequency of alternate allele). The red, green and yellow bars on the left indicate the haplotype on which the detected variation resides (risk  =  red; protective  =  green; other haplotypes  =  yellow). Detected variation with respect to the specific haplotype on which it resides is shown to the right of haplotype bars, where non-reference alleles are coloured in light blue (homozygous) and dark blue (heterozygous). The locations of the six SNP markers that were used to define the haplotypes are shown below the variation. The location of the PreSTIGE enhancer elements within the 454 sequenced region from human skeletal muscle myoblasts (HSMM) are indicated in orange, and from neuronal precursor cells (NPC) are indicated in purple. PreSTIGE enhancer locations were obtained from <a href="http://genetics.case.edu/prestige/" target="_blank">http://genetics.case.edu/prestige/</a>).</p

Heatmap of mean depth of coverage per amplicon and per sample.

<p>Amplicons are listed along the x-axis, and samples (x1-48) are listed along the y-axis. Depth of coverage is indicated according to the colour key, with white indicating <2 reads coverage and red indicating >35 reads coverage. The plot was created using R <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092556#pone.0092556-R1" target="_blank">[63]</a>.</p

Region of <i>NCAM1</i> gene covered via Roche 454 sequencing.

<p>The sequenced region spanned 6.6(chr11∶113,100,972–113,107,571 bp; hg19) and included exons 8–12 (red boxes) and less frequently used exons 8a and mini-a (grey boxes) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092556#pone.0092556-Atz1" target="_blank">[52]</a>. The locations of the 5^th and 6^th glycosylation sites and acidic patch required for glycosylation are in exons 9, 10 and 11 and are indicated by green, blue and white lines, respectively. The locations of SNPs identified in this study are indicated with arrows. Names of known SNPs are in black text, and novel SNPs are listed as NV (novel base substitutions) or InDel (novel insertion-deletion change) followed by their base position (hg19). Two coding SNPs located in exons 8a and 11 are marked with closed boxes, with their effect on amino acid sequence shown.</p

Summary of genetic variation identified in NCAM1.

<p>The location of each variant (NCBI 37/hg19 build) relative to known exons is shown, with exon locations indicated, and coding SNPs indicated with asterisks. The minor allele frequency (MAF) of each variant in all samples (BP+1 kG) and the BP and 1 kG cohorts separately is given. The frequency difference (freqDIFF) was calculated relative to 1 kG allele frequency, with absolute frequency differences greater than 5% indicated in bold text, and those with p values <0.1 indicated with an asterisk.</p>a<p>For each polymorphism, the minor allele is listed first.</p>v<p>SNPs verified by direct genotyping are indicated.</p

Summary of transcribed and DHSP overlapping genetic variation identified in <i>ST8SIA2</i>.

<p>The location of transcribed and DHSP overlapping variants on chromosome 15 (hg19 build), with transcribed SNPs indicated with asterisks. SNP alleles (mA) identified exclusively on the risk haplotype are shown (0 =  not exclusive, 1 =  present on risk and other haplotypes, 2 =  present on risk haplotype only, ND  =  not determined). SNPs identified exclusively on the protective haplotype are shown (0 =  not exclusive, 1 =  present on protective and other haplotypes, 2 =  present on protective haplotype only, ND = not determined). The set from which the SNP was observed is given (1 =  GATK & Refmapper; 2 =  GATK only; 3a  =  Refmapper only; 3b  =  Refmapper only & filtered in GATK; 4 =  Sanger). Co-localisation with DNase I hypersensitivity site (DHS) peaks (neuronal = 1; hESC = 2; foetal brain = 3) are given. Genomic Evolutionary Rate Profiling (GERP) scores are provided for each variant that is within a GERP-conserved element. The nature of the polymorphism in each cohort is given (with minor allele listed first). The minor allele frequency (MAF) of each variant in the 47 bipolar cases and 174 Caucasian individuals (CEU and GBR) from the 1000 Genomes Project (1 kG) are shown separately. The frequency difference (freqDIFF) was calculated relative to 1 kG allele frequency, with absolute frequency differences greater than 5% indicated in bold text, and those with p values <0.1 indicated with an asterisk.</p>a<p>For each polymorphism, the minor allele is listed first.</p>v<p>SNPs verified by direct genotyping are indicated. The annotated list of all <i>ST8SIA2</i> variation identified is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092556#pone.0092556.s003" target="_blank">Table S1</a>.</p

Coverage of <i>ST8SIA2</i> gene locus by 454 and Sanger sequencing.

<p>The 96∼20 kb flanking sequence (chr15∶92,919,255–93,013,920 bp; UCSC Genome Browser hg19 <a href="http://genome.ucsc.edu" target="_blank">http://genome.ucsc.edu</a>) was divided into eight long-range PCR amplicons (454 Amplicons 1–8, in pink) for library preparation prior to 454 sequencing. The mean depth of coverage across all 48 samples for each long-range amplicon is shown in burgundy. A GC-rich region surrounding the promoter and exon 1 was sequenced via Sanger sequencing using 8 short overlapping amplicons (Sanger amplicons 1–8; inset). Pink dots on the GC percent or mean depth per sample tracks indicate loci where the level of GC richness, or read depth is above the range indicated. Regions which failed to amplify, or which contained simple repeats, long homopolymers (length > = 10), or short interspersed elements (SINEs) tended to have lower depth of coverage, and were inaccessible to next-generation sequencing in phase I of the 1000 genomes project (1000 G Accs strict).</p

Fourteen novel SNPs assayed by Sequenom for verification.

<p>Three SNPs that were detected in 454-generated sequence were non-polymorphic (NP) after direct genotyping. The allele count (AC) is given from a total of 380 subjects (215 cases including the 48 sequenced individuals, 165 controls). The algorithm set from which the SNP was observed is provided (1 =  GATK & Refmapper; 2 =  GATK only; 3a  =  Refmapper only; 3b  =  Refmapper only & filtered in GATK; 4 =  Sanger).</p