Damara Hamlin

ASSOCIATION OF GENETIC VARIANTS IN INTRON 2 OF THE NEUREXIN 1 GENE WITH ALCOHOL DEPENDENCE AND NICOTINE DEPENDENCE. Damara Hamlin¹, Tony Hinrichs², Jen C. Wang², Laura Bierut², Allison Goate², Biology Department, Washington University in St. Louis, MO¹; Psychiatry Department, Washington University Medical School, St. Louis, MO².

Alcoholism and nicotine dependence show moderate heritability and share common genetic risk factors. The Collaborative Study on the Genetics of Alcoholism (COGA project) is a multicenter family study funded by the National Institute on Alcohol Abuse and Alcoholism with the goal of identifying genes that affect the risk for alcoholism and other related phenotypes. One gene of interest is Neurexin 1 (NRXN1), which is mapped to chromosome 2p16. NRXN1 is one of the largest genes in the human genome; it contains 24 exons and spans 1.1 Mb. NRXN1 belongs to a family of proteins that behave as cell adhesion molecules and receptors in the vertebrate nervous system. In a genetic mapping study using COGA data, alcohol dependence related haplotypes were identified within NRXN1, yielding and odds ratio of 0.65 (Yang et al., 2005). Also, unpublished data from Bierut et al. in a genome-wide association analysis shows that NRXN1 may be associated with smoking phenotypes.

We analyzed the NRXN1 gene to determine whether variation in this gene predisposes individuals to alcoholism and smoking behaviors. We focused on intron 2 of the NRXN gene, an area that has known SNPs of interest from Dr. Bierut’s study. Six SNPs were genotyped in the COGA sample and then tested for association with COGA alcohol dependence, DSM4 alcohol dependence, ICD10 alcohol dependence, smoking phenotypes, and nicotine addiction. MassARRAY spectrometry (Sequenom, San Diego, CA, USA) methods were used for SNP genotyping. PCR primers, termination mixes, and multiplexing capabilities were determined with Sequenom Spectro Designer software v2.00.17. Standard PCR procedures were used to amplify PCR products. All unincorporated nucleotides were deactivated with shrimp alkaline phosphatase. A primer extension reaction was then carried out with the mass extension primer and the appropriate termination mix. The primer extension products were then cleaned with resin and spotted onto a silicon SpectroChip. The chip was scanned with a mass spectrometry workstation (Bruker) and the resulting genotype spectra were analyzed with the Sequenom SpectroTYPER software. PEDCHECK was used to check for Mendelian errors in the data, and scores for unresolved errors were zeroed out of the file before analysis. All SNPs were tested to check that they are in Hardy Weinberg Equilibrium in the founders of our dataset in Caucasian and African American families separately. Finally, the programs Transmit and PDTPHASE were used to test for association between variants of NRXN1 and alcohol dependence and smoking phenotypes.

Of the six SNPs analyzed, we found one that showed association with the three different measures of alcohol dependence as well as the model of nicotine addiction. Future directions may consider genotyping coding SNPs in this gene and SNPs within and flanking this associated LD block. Also, the SNPs could be tested for association with other phenotypes, such as models of depression.

References

1. Yang, H. et al. (2005). “A genome-wide scanning and fine mapping study of COGA data.” BMC Genetics 6(Suppl I): S30