A novel method for high throughput genotyping of copy number variation (CNV) has been developed by Dr Philip Guthrie within Bristol Genetic Epidemiology Laboratories. This has been optimised for two CNVs: haptoglobin and salivary amylase. This project aims to genotype these two polymorphisms in the ALSPAC cohort.

Haptoglobin (HP) copy number

Haptoglobin acts as an antioxidant by limiting peroxidative tissue damage by free hemoglobin, which it specifically binds with high affinity. It has also been reported to associate with vitamin C level. Haptoglobin allele Hp2 comprises a 1.7kb duplication encompassing exons 3 and 4, relative to allele Hp1. Hp2 carriers form protein multimers which have reduced availability to tissues, and have been claimed to be associated with higher rates of diabetic vascular complications. Most genetic studies have been conducted by serum phenotyping since there remains a shortfall of DNA-based assays of the duplicon structure.

The only known qualitative difference of allele Hp2 from Hp1 is the presence of the junction sequence between the duplicated segments. We have developed a liquid-phase assay for this junction which is simple, robust, economical and high throughput, and we have applied it to analyse the BWHHS (female) and CAPS (male) cohorts. These studies revealed apparently gender-specific associations. For example, BWHHS showed associations between Hp genotype and haemoglobin level, erythrocyte count and coronary heart disease, whereas Caerphilly showed only a non-significant trend for haemoglobin level in the same direction as in BWHHS.

Analysis of the ALSPAC cohort for Hp genotype will greatly increase the power of this study and help to elucidate further the roles of sex difference in modulating the effects of genotype.

This assay would typically require 10ng genomic DNA. DNA from cell lines is unsuitable, as it may have additional variation in copy number not present in the original sample. We would, however, like to carry out a pilot study using e.g. 100-200 samples replicated between genomic and cell line DNA, to verify/quantify this phenomenon.

Salivary Amylase (AMY1) copy number

Amylase is the enzyme responsible for starch hydrolysis, and the salivary amylase gene AMY1 shows extensive variation in diploid copy number, varying from 2 to 16 copies. A recent study concluded that the pattern of variation in copy number of this gene is consistent with diet-related selection pressures. We have generated an additional hypothesis, which is that copy number of this gene is likely to be more strongly associated with selection pressures generated from interaction between the amount of gene product and some species of Streptococci, which capture amylase onto their coats for their own ends; this in turn influences which other pathogens can colonise or infect the mouth/pharynx, with possible disease-related outcomes due to competition between Streptococci and other buccal micro-organisms

We have developed a liquid-phase assay to determine copy number of this gene, which is simple, robust, economical and high throughput. This assay would typically require 10ng genomic DNA. DNA from cell lines is unsuitable, as it may have additional variation in copy number not present in the original sample. We would like to carry out a pilot study using e.g. 100-200 samples replicated between genomic and cell line DNA, to verify/quantify this phenomenon.