The main aim of our study was to detect those SNPs and genes previously associated with alcoholism that could be replicated in our subgroup of alcoholic bipolar research subjects. Demographic breakdown of the groups was as follows: i) BPALC male 80 (56%), female 63 (44%). ii) NABPD, male 113 (31%), female 250 (69%). iii) Controls male 217 (43%), female 293 (57%).
There were 3799 SNPs nominally associated with alcoholic bipolar subjects with significance p <0.01. 2136 of these SNPs were within 50kb of a gene and 1491 were within the gene. The nominally significant associations are reported in and Supplementary Table 1
. Twelve SNPs attained p-values of <1.0 × 10−4
but no markers attained conventional genome-wide significance levels. lists the tests of association with SNPs associated with bipolar alcoholism and in the non alcoholic bipolar group (NABPD) and this enables us to differentiate genetic effects on bipolar alcoholism from genetic effects on pure bipolar disorder (ie NABPD). Supplementary Table 1
reports the full genome wide findings, arranged by BPALC p-value, where SNPs had p<0.01.
Genome wide association results for positive SNPs in bipolar alcoholism (BPALC) and in non-alcoholic bipolar disorder (NABPD) compared with controls
As described in the methods section, in order to confirm the involvement of genes previously implicated in alcoholism and addictions phenotypes, we employed gene wise permutation tests on all SNPs within and next to a previously implicated gene and corrected for multiple tests . The results from these tests are shown in . Replications with gene wise significance at p<0.05 for association with the bipolar alcoholic group included the cadherin 11 (CDH11), collagen type XI alpha 2 (COL11A2), neuromedin U receptor 2 (NMUR2), exportin7 (XP07) and semaphorin associated protein 5A (SEMA5A) genes. Only the gene wise association with CDH11 remained significant after Bonferroni correction for the genes tested (n=38). Also shown in are the tests of association with SNPs in the alcoholism associated genes in the bipolar research subjects after the alcoholic bipolar subjects have been removed, denoted once again as the NABPD group.
Table 2 Gene-wise permutation tests of association for genes both i) implicated by SNP markers for bipolar alcoholism (BPALC) and ii) replicating associations with alcoholism and related phenotypes; contrasted with SNP and gene wise data in non-alcoholic bipolar (more ...)
Lastly, genes which were implicated by SNP associations and replication, but which did not achieve gene-wise significance in our sample are also detailed in . The most replicated alcoholism genes ALDH, ADH and GABRA2 were not implicated at SNP or gene wise level in the BPALC phenotype.
We also note that the remaining SNPs which we found were nominally associated with BPALC and which confirmed previous reported gene associations with addiction phenotypes in the “connectivity constellation” failed to show gene-wise significance in our study (data not shown). These connectivity genes were: the neural cell adhesion molecule (NRCAM
) gene, brain-specific angiogenesis inhibitor 3 (BAI3
), contactin associated protein-like 2 (CNTNAP2
), CUB and Sushi multiple domains 1 (CSMD1
), catenin (cadherin-associated protein), alpha 2 and 3 (CTNNA2 & 3
), Down syndrome cell adhesion molecule (DSCAM
), protein tyrosine phosphatase, receptor type, D (PTPRD
), sarcoglycan zeta (SGCZ
), brain-specific angiogenesis inhibitor 3 (BAI3
), astrotactin 2 (ASTN2
), ankyrin repeat and sterile alpha motif domain containing 1B (ANKS1B
), contactin 4 and 6 (CNTN4 & 6
) (Uhl et al., 2008b
). The SNPs associated with these genes are listed in Supplementary Table 1
The top-ranked (by genome wide significance values) alcoholic bipolar SNP associations and the genes they represent are listed in and continued in Supplementary Table 1
. Selected findings are noted below. The most significant SNP (p=4.54×10−6
) was rs8062326, a single SNP placed midway between the synaptotagmin 17 (SYT17
) and Inositol 1,4,5-triphosphate receptor-interacting protein-like 2 (ITPRIPL2
) genes. One of two SNPs intronic to the collagen, type I, alpha 2 (COL1A2
, highest ranked SNP rs369982, p = 2.06 × 10−5
) gene was ranked second most associated and when all SNPs in this gene were analysed the gene-wise test was significant at p = 5 × 10−4
. Ranked 29th, the first of forty five SNPs nominally associated with BPALC were located in and around cadherin 11 (CDH11
, highest ranked SNP rs429065, p = 1.0 × 10−4
, gene-wise p = 6 × 10−4
). rs429065 is located 22 Kb downstream of CDH11
(). Eleven intronic SNPs at CDH11
were found to be associated with BPALC of which two were also nominally associated with NABPD. Two of the associated SNPs from a genome-wide study of pooled DNA from the COGA sample (Johnson et al., 2006
) were also nominally associated in our sample. These were intronic SNP rs35200 (p = 1.9 × 10−2
) and rs35164 (p = 1.4 × 10−4
) which is 5,484 base pairs downstream from CDH11
. rs25164 was also implicated with early age of onset alcoholism in the GWAS by Edenberg and colleagues (Edenberg et al., 2010
). Excluding the CDH11 SNPs replicated above, no strongly associated SNPs from previous GWAS replicated in this study. Eighteen SNPs within the introns of cadherin 12 (CDH12
, ) and 10 of 14 SNPs genotyped within the introns of cadherin 13 (CDH13,
) were nominally associated with BPALC. In total eighty four SNPs in or near cadherin-family genes were nominally associated with BPALC.
Figure 1 The CDH11 SNP (rs429065) with the most significant BPALC association is shown (large red diamond) with its P-value. Estimated recombination rates (taken from HapMap) are plotted to reflect LD structure. LD between rs429065 and the other SNPs genotyped (more ...)
Novel genes, implicated by clustering of 7 or more SNPs (p<0.01) and associated on gene wise testing in the bipolar alcoholic sample
and Supplementary Table 1
also report the top-ranked BPALC-associated SNPs which were also nominally significant in NABPD genetic associations. Most significant and ranked 2nd
genome wide was a SNP intronic to musashi homolog 2 (MSI2
, highest ranked SNP rs1024820, p = 1.24 × 10−5
). However these SNPs were not in LD with the MSI2
gene, nor was the gene associated with bipolar alcoholism using gene-wise analysis. There were six BPALC associated SNPs in or within 25 Kb of tetraspanin 8 (TSPAN8
, highest ranked SNP rs1705236, p = 1.24 × 10-4; gene wise p non significant) three of which were also associated with NABPD.
We found six nominally BPALC-associated SNPs near or within the GABA-A beta-3 receptor (GABRB3, highest ranked SNP p = 2.6 × 10−4, uncorrected gene-wise significance p = 2 × 10−3). The tachykinin receptor 1 (TACR1, also known as neurokinin-1 receptor, NK1R) was nominally associated with BPALC with three SNPs, one of which, rs3771829 (p = 3 × 10−3) was also associated with NABPD (p = 1.7 × 10−2). There were eight SNPs nominally associated with BPALC near the gene for insulin-like growth factor 1 (IGF1, highest ranked SNP: rs12426318, 154 kb downstream, p = 1.8 × 10−3). Neither TACR1 nor IGF1 survived gene wise testing for either phenotype.
Lastly we performed gene wise testing on the remaining genes implicated by clustered (7 or more) nominal SNP associations with the BPALC phenotype, but which had no prior literature association with alcoholism or related phenotypes. These represent novel BPALC candidate genes (). Three genes survived Bonferroni correction: KIAA1772, DC2 protein, and zinc finger and BTB domain containing 16 (ZBTB16).